KR20210089648A - Methods and compositions for messenger RNA purification - Google Patents

Abstract

In particular, the present invention relates to an in vitro transcription process (IVT)-synthesized mRNA by precipitating it in a buffer containing a denaturing salt together with a reducing agent, then capturing the precipitated mRNA, and dissolving the captured mRNA into a solution to obtain purified mRNA. By obtaining, a method for the purification of mRNA is provided, which involves removing impurities from a messenger RNA preparation synthesized by a large-scale in vitro transcription process (IVT).

Description

Methods and compositions for messenger RNA purification

<Related Application>

This application claims priority to U.S. Provisional Application Serial No. 62/757,612, filed November 8, 2018, the disclosure of which is incorporated herein by reference.

Messenger RNA therapy (MRT) is a promising new approach to treat a variety of diseases. MRT involves administering messenger RNA (mRNA) to a patient in need of therapy. The administered mRNA produces a protein or peptide encoded by the mRNA within the patient's body. mRNA is typically synthesized using an in vitro transcription system (IVT) that involves an enzymatic reaction by RNA polymerase. The IVT synthesis process is usually followed by reaction(s) for the addition of a 5'-cap (capping reaction) and a 3'-poly A tail (polyadenylation). These reactions include not only the full-length mRNA thus formed, but also various undesirable contaminants in the resulting reaction mixture (e.g., proteins, salts, buffers, and non-mRNA nucleic acids used or produced in the reaction). resulting in a composition that A variety of purification methods can be used to remove contaminants to provide purified mRNA. However, trace amounts of residual contaminant proteins or enzymes can lead to unknown side effects in patients. To achieve purified mRNA suitable for administration in a subject, several rounds of purification are currently required. Thus, conventional purification methods and compositions are inadequate to provide clean, homogeneous mRNA suitable for patient administration and free of contaminants without costly multi-step purification processes.

There is a need for new and improved methods to achieve mRNA for therapeutic use that is free of contaminants, particularly residual proteins or enzymes from the mRNA synthesis process.

The present invention provides improved purification methods for mRNA synthesized in vitro. The present invention is based, in part, on the surprising discovery that precipitation of mRNA in a buffer comprising high concentrations of guanidinium salt and detergent greatly improves the removal of protein contaminants from the mRNA product. Specifically, a single precipitation of capped and tailed mRNA followed by a purification step can successfully remove detectable levels of residual enzyme from in vitro synthesized mRNA. Reducing the number of purification steps (eg, repeated precipitation steps) results in an easier and more cost effective fabrication process. More significantly, the compositions and methods described herein produce unexpectedly high mRNA recovery, purity and integrity. Thus, the present invention allows for more efficient and cost-effective production of mRNA for therapeutic use. Additionally, the method of the present invention is useful for purifying large amounts of mRNA.

In one aspect, the present invention is a method for removing impurities from a messenger RNA (mRNA) preparation synthesized on a large scale by an in vitro transcription process (IVT), wherein IVT-synthesized mRNA is combined with a reducing agent as a denaturing salt (denaturing salt). ) precipitating in a buffer containing; capturing the precipitated mRNA; and dissolving the captured mRNA into a solution to purify the mRNA. In some embodiments, the method further comprises capping and tailing the mRNA prior to precipitating the mRNA. In some embodiments, impurities in an mRNA preparation obtained by a large-scale IVT process include proteins (including enzymes used for IVT, capping and tailing); incomplete RNA transcript; nucleotides and salts.

In some embodiments, the purified mRNA obtained by the method is greater than at least 90% of the amount of synthesized mRNA.

In some embodiments, the purified mRNA obtained by the method is substantially free of any enzymatic impurities.

In some embodiments, at least about 1 mg of mRNA is synthesized in the IVT reaction. In some embodiments, at least about 50 mg of mRNA is synthesized in the IVT reaction. In some embodiments, at least about 1 gram (G) of mRNA is synthesized in an IVT reaction. In some embodiments, at least about 10 G of mRNA is synthesized in an IVT reaction. In some embodiments, at least about 100 G of mRNA is synthesized in an IVT reaction. In some embodiments, at least about 1 Kg of mRNA is synthesized in an IVT reaction.

In some embodiments, the buffer for the precipitation step further comprises an alcohol. In some embodiments, the precipitation step is performed under conditions wherein mRNA, denaturing buffer and alcohol are present in a volume ratio of 1: (5): (3). In some embodiments, the precipitation step is performed under conditions wherein the mRNA, denaturing buffer and alcohol are present in a volume ratio of 1: (3.5): (2.1). In some embodiments, the precipitation step is performed under conditions wherein mRNA, denaturing buffer and alcohol are present in a volume ratio of 1: (2.8): (1.9). In some embodiments, the precipitation step is performed under conditions wherein mRNA, denaturing buffer and alcohol are present in a volume ratio of 1: (2.3): (1.7). In some embodiments, the precipitation step is performed under conditions wherein the mRNA, denaturing buffer and alcohol are present in a volume ratio of 1: (2.1): (1.5).

In some embodiments, the modified salt is guanidinium thiocyanate. In some embodiments, the denaturing buffer comprises guanidine thiocyanate-sodium citrate, N-lauryl sarcosine (GSCN).

In some embodiments, the guanidinium thiocyanate is present at a final concentration of at least 2 M, at least 3 M, at least 4 M, at least 5 M, at least 6 M, or at least 7 M. In some embodiments, the guanidinium thiocyanate is present in a final concentration of at least greater than 3 M. In some embodiments, the guanidinium thiocyanate is present in a final concentration of at least greater than 4 M. In some embodiments, the guanidinium thiocyanate is present at a final concentration of 5 M.

In some embodiments, the reducing agent is dithiothreitol (DTT), beta-mercaptoethanol (β-ME), tris(2-carboxyethyl)phosphine (TCEP), tris(3-hydroxypropyl)phosphine ( THPP) and dithiobutylamine (DTBA). In some embodiments, the reducing agent is dithiothreitol (DTT).

In some embodiments, DTT is present at a final concentration greater than 1 mM and up to about 200 mM. In some embodiments, DTT is present at a final concentration of 2.5 mM to 100 mM. In some embodiments, DTT is present at a final concentration of 5 mM to 50 mM. In some embodiments, the concentration of DTT is 5 mM, 10 mM, 15 mM or 20 mM.

In some embodiments, the step of precipitating the mRNA is performed more than once. In some embodiments, the step of precipitating the mRNA is performed only once.

In some embodiments, capturing the mRNA further comprises performing filtration, centrifugation, reversible adsorption to a solid phase, or a combination thereof on the mRNA.

In some embodiments, the filtration is tangential flow filtration (TFF).

In some embodiments, the filtration is depth filtration (DF).

In some embodiments, the filtration is performed using a Nutsche filter.

In some embodiments, filtration is performed in conjunction with centrifugation.

In some embodiments, the step of lysing the captured mRNA is performed in an aqueous solution.

In some embodiments, the stage of captured mRNA is subjected to dialysis in a suitable buffer comprising a lower salt concentration.

In some embodiments, the purified composition is substantially free of any protein impurities, which are less than 5%, less than 4%, less than 3%, less than 2%, less than 1% protein impurities, or 0% protein impurities.

In some embodiments, the purified mRNA is substantially free of salts.

In some embodiments, the purified mRNA comprises at least greater than 95%, greater than 96%, greater than 97%, greater than 98%, greater than 99%, or about 100% of the full length mRNA encoding the protein.

The patents and scientific literature referred to herein establish the knowledge available to those skilled in the art. All US patents and published or unpublished US patent applications cited herein are incorporated by reference. All published foreign patents and patent applications cited herein are incorporated herein by reference. All other published references, documents, manuscripts and scientific literature cited herein are incorporated herein by reference.

Other features and advantages of the present invention will be apparent from the drawings and the following detailed description and claims.

These and further features will become more apparent from the following detailed description when considered in conjunction with the accompanying drawings. However, the drawings are for illustrative purposes only and not limiting.
1 depicts a silver stained gel analysis of purified RNase 1 digested product for detection of residual protein in mRNA preparations. CFTR mRNA was purified by column filtration. Lane 1 of gel 1 (left image) or gel 2 (right image) corresponds to the positive control mRNA showing a hazy band of contaminant proteins. gel 1, lane 5 shows a sample from purification using 4 M guanidine thiocyanate-sodium citrate, N-lauryl sarcosine (GSCN) buffer; gel 2, lane 5 shows a sample from purification using 5 M GSCN-1% lauryl sarcosine buffer; Gel 2, lane 6 shows a sample from purification using 5 M GSCN-0.1% lauryl sarcosine buffer. Lanes 9-11 of either gel depict electrophoretic transfer controls for RNase 1, SP6 and guanylate transferase, respectively. Other lanes not identified herein are out of context (n/a). Arrows indicate positions for migration of major contaminant bands, SP6 polymerase/guanylate transferase.
2 depicts a silver stained gel analysis of purified RNase 1 digested mRNA products for detection of residual protein in mRNA preparations. CFTR mRNA was purified by filtration in 24-well microtiter plates using guanidine thiocyanate buffer. Lane 1 of gel 1 (left image) or gel 2 (right image) corresponds to the positive control mRNA showing a hazy band of contaminant proteins. gel 1, lane 6 shows a sample purified using 5 M guanidine thiocyanate buffer without DTT; gel 2, lane 6 shows a sample purified using 5 M guanidine thiocyanate buffer without DTT; Lanes 10-12 of either gel depict electrophoretic transfer controls for RNase 1, SP6 and guanylate transferase, respectively. Other lanes not identified herein are out of context (n/a). Arrows indicate migration of the main contaminant band, SP6 polymerase/guanylate transferase.
3 depicts a silver stained gel analysis of a gram-scale purified and RNase 1 digested product for detection of residual protein in mRNA preparations. Lane 1 shows a blurry positive control sample. Lane 5 shows a sample from purification of 1 g of CFTR mRNA preparation using 5 M GSCN w/ DTT buffer. Lanes 6-10 depict electrophoretic transfer controls for RNase 1, SP6, guanylate transferase, poly A polymerase and O-methyl transferase, respectively. Other lanes not identified herein are out of context (n/a). Arrows indicate positions for migration of major contaminant bands, SP6 polymerase/guanylate transferase.
4 depicts a silver stained gel analysis of a 10 gram (10 G)-scale purified RNase 1 digested product for detection of residual protein in mRNA preparations. Lane 5 shows a sample from purification of 10 g of CFTR mRNA preparation using 5 M GSCN w/ DTT buffer. Lanes 7-11 depict electrophoretic transfer controls for RNase 1, SP6, guanylate transferase, poly A polymerase and O-methyl transferase, respectively. Arrows indicate positions for migration of major contaminant bands, SP6 polymerase/guanylate transferase. Other lanes not identified herein are out of context (n/a).
5A-B depict capillary electrophoresis of 10 G-scale purified mRNA samples. Figure 5A, mRNA product without tailing; Figure 5B, after tailing reaction.
6 depicts a silver stained gel analysis of 100 gram (100 G)-scale purified RNase 1 digested product for detection of residual protein in mRNA preparations. Lanes 5 and 6 show the purified product. Lane 1 depicts a control purified mRNA sample showing a faint band of contaminant enzyme. Lanes 8-10 depict electrophoretic transfer controls for the process enzymes RNAse1, SP6, guanylate transferase, poly A polymerase and 2-O-methyl transferase, respectively.
7 depicts capillary electrophoresis of 100 G-scale purified mRNA samples. Peaks indicated by curved arrows represent mRNA purified by the method of the present invention. Peaks shown with straight arrows represent control samples treated with 4 M GSCN buffer.
8 depicts purified silver stained gel assay mRNA products with various reducing agents. Lane 2 shows the purified product using only 5 M GSCN without reducing agent. Lanes 3-9 show the purified product using 5 M GSCN with various reducing agents at different concentrations. Lanes 10-12 depict electrophoretic transfer controls for the process enzymes RNase I, SP6 and guanylate transferase, respectively.

<Definition>

In order to more readily understand the present invention, certain terms are first defined below. Additional definitions for the following terms and other terms are set forth throughout the specification.

As used in this specification and the appended claims, singular expressions include plural referents unless the context clearly dictates otherwise.

As used herein, the term “or” is understood to be inclusive and covers both “or” and “and” unless specifically stated or clear from the context.

As used herein, the terms “for example” and “i.e.,” are used by way of example only, and not intended to be limiting, and should not be construed as referring only to those items explicitly recited in the specification.

Throughout the specification the word "comprising" or variations such as "comprises" or "comprising" is understood to mean including the stated element, integer or step, or group of elements, integers or steps. but does not exclude any other element, integer or step, or group of elements, integers or steps.

Unless specifically stated or clear from context, as used herein, the term “about” is to be understood as within the normal tolerance of the art, eg, as within 2 standard deviations of the mean. “About” means 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, 0.1%, 0.05%, 0.01% or It can be understood to be within 0.001%. Unless otherwise clear from context, all numerical values provided herein are modified by the term “about.”

As used herein, the term “batch” refers to an amount or quantity of mRNA synthesized at one time (eg, produced according to a single production sequence during the same production cycle). A batch may refer to the amount of mRNA synthesized in one reaction that occurs via a single aliquot of an enzyme and/or a single aliquot of a DNA template in the case of continuous synthesis under a set of conditions. In some embodiments, a batch will include mRNA resulting from a reaction wherein not all reagents and/or components are replenished and/or replenished as the reaction proceeds. The term “batch” shall not refer to mRNA synthesized at different times that are combined to achieve a desired amount.

As used herein, the term “contaminant” refers to a substance within a defined amount of a liquid, gas, or solid, which differs from the chemical composition of the target material or compound. Contaminants are also referred to as impurities. Examples of contaminants or impurities include buffers, proteins (eg, enzymes), nucleic acids, salts, solvents, and/or wash solutions.

As used herein, the term “dispersant” refers to a solid particulate that reduces the likelihood that mRNA precipitates will form a hydrogel. Examples of dispersants include one or more of ash, clay, diatomaceous earth, filter media, glass beads, plastic beads, polymers, polypropylene beads, polystyrene beads, salts (eg, cellulose salts), sand, and sugar. and is not limited thereto. In an embodiment, the dispersant is polymeric microspheres (eg, poly(styrene-co)-divinylbenzene) microspheres.

As used herein, "expression" of a nucleic acid sequence refers to one or more of the following events: (1) generation of an mRNA template from a DNA sequence (eg, by transcription); (2) processing of mRNA transcripts (eg, by splicing, editing, 5' cap formation and/or 3' end formation); (3) translation of mRNA into a polypeptide or protein; and/or (4) post-translational modifications of the polypeptide or protein. In this application, the terms "expression" and "generating" and grammatical synonyms are used interchangeably.

As used herein, "full length mRNA" is characterized when using detection using UV and UV absorption spectroscopy in conjunction with separation by specific assays, such as gel electrophoresis, or capillary electrophoresis. The length of the mRNA molecule as obtained according to any of the purification methods described herein and encoding the full-length polypeptide is at least 50% of the length of the full-length mRNA molecule transcribed from the target DNA, e.g., any of the methods described herein. At least 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97% of the length of the full-length mRNA molecule transcribed from the target DNA and prior to purification according to the method , 98%, 99%, 99.01%, 99.05%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%.

As used herein, the term "isolated" means that (1) when initially created it has been separated from at least some of its associated components (naturally and/or in experimental settings) and/or (2) by human hands. to a substance and/or entity created, manufactured and/or manufactured by

As used herein, the term “messenger RNA (mRNA)” refers to a polyribonucleotide encoding at least one polypeptide. mRNA as used herein encompasses both modified and unmodified mRNA. An mRNA may contain one or more coding and non-coding regions. mRNA may be purified from natural sources, produced using recombinant expression systems, and optionally purified, transcribed in vitro, or chemically synthesized.

As used herein, the term “mRNA integrity” generally refers to the quality of mRNA. In some embodiments, mRNA integrity refers to the percentage of mRNA that is not degraded after a purification process (eg, a method described herein). mRNA integrity can be determined using methods specifically described herein, such as TAE agarose gel electrophoresis, or by SDS-PAGE with silver staining, or by methods well known in the art, for example RNA agarose. gel electrophoresis (eg, Ausubel et al., John Wiley & Sons, Inc., 1997, Current Protocols in Molecular Biology).

As used herein, the term "pharmaceutically acceptable" means, within the scope of sound medical judgment, suitable for use in contact with tissues of humans and animals without undue toxicity, irritation, allergic reaction, or other problems or complications, and , which is compatible with a reasonable benefit/risk ratio.

"Pharmaceutically acceptable excipient" means an excipient suitable for the manufacture of a pharmaceutical composition that is generally safe, non-toxic, and not biologically or otherwise undesirable, which is acceptable for veterinary as well as human pharmaceutical use. including excipients. "Pharmaceutically acceptable excipient" as used in the specification and claims includes both one and more than one such excipient.

Typically, suitable mRNA solutions may also contain buffers and/or salts. In general, buffers may include HEPES, ammonium sulfate, sodium bicarbonate, sodium citrate, sodium acetate, potassium phosphate and sodium phosphate.

As used herein, the term “substantially” refers to a qualitative condition that exhibits the full or nearly full extent or degree of a characteristic or characteristic of interest. Those of ordinary skill in the art of biology will understand that it is rare (if any) that biological and chemical phenomena progress to perfection and/or perfection, or achieve or avoid absolute results. Thus, the term “substantially” is used herein to capture the potential lack of integrity inherent in many biological and chemical phenomena. Accordingly, a composition substantially free of compound 'x' will be understood to include less than 5% compound 'x', or less than 1% or less than 0.1% or less than 0.01% compound 'x'. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly used in the art to which this application belongs and as commonly understood by one of ordinary skill in the art to which this application belongs; Such journals are incorporated by reference in their entirety. In case of inconsistency, the present specification, including definitions, will control.

<Detailed description>

The present invention relates to methods for preparing scalable amounts of pure and high quality mRNA. mRNA is typically synthesized by in vitro transcription (IVT) using a polymerase such as SP6 or T7-polymerase, and then capped and tailed to generate full-length in vivo translatable mRNA. However, proteins associated with mRNA synthesis and/or capping and tailing reactions (“process enzymes”) are difficult to remove from the mRNA product using conventional purification methods. As a result, purification processes often involve multiple precipitation steps to isolate mRNA from contaminant proteins. Because mRNA is structurally unstable and prone to agitation-related degradation, repeated precipitation, centrifugation and/or filtration steps are likely to affect mRNA integrity, which is undesirable for downstream therapeutic applications.

The present invention provides, at least in part, a significant improvement in the removal of contaminating proteins when a buffer comprising a higher than usual concentration of a guanidine salt (eg, guanidine thiocyanate) is used for mRNA precipitation and processing. It is based on the surprising and unexpected discovery that it is observed. Additionally, in some embodiments, when the buffer comprising a high concentration of guanidine salt further comprises a detergent as described herein, the buffer for precipitation is added to the buffer for precipitation without detergent in the removal of protein contaminants from the mRNA preparation. shows a higher efficiency than In some embodiments, the buffer comprises guanidine thiocyanate-sodium citrate, N-lauryl sarcosine (GSCN). Guanidine thiocyanate and GSCN are often used interchangeably throughout applications in connection with denaturing buffers. In some embodiments, using the buffers and methods of the invention as described, high quality purified mRNA is produced after a single precipitation step. In some embodiments, the term “process enzyme” includes enzymes, or proteins, or protein fragments thereof. In some embodiments, process enzymes may include, but are not limited to, polymerases, or phosphatases, or kinases, or guanylyl transferases, or other proteins, or fragments thereof. In some embodiments, the term protein contaminant as used herein may include contaminating enzymes, proteins or protein fragments thereof, which may include polypeptides or oligopeptides (peptides).

In addition, the buffers and methods of the present invention are scalable to produce large quantities of purified mRNA in batches. Thus, the methods and compositions of the present invention provide advantages in reducing the number of steps in a large scale fabrication process.

In some embodiments, large-scale purification comprises at least 1 gram mRNA, such as 5 grams or more, 10 grams or more, 20 grams or more, 50 grams or more, 100 grams or more, 150 grams or more, 200 grams or more, 500 grams or more, 1000 grams or more. , or purifying a composition comprising at least 10,000 grams of mRNA. In some embodiments, the large scale mRNA synthesis process comprises greater than about 1 gram of mRNA, or greater than about 10 grams of mRNA, or greater than about 20 grams, or greater than about 50 grams, or greater than about 100 grams, or greater than about 150 grams; or greater than about 200 grams, or greater than about 500 grams, or greater than about 1000 grams, or greater than about 10,000 grams of mRNA.

messenger RNA

The purification methods described herein are suitable for the purification of any mRNA. The present invention can be used to purify mRNA encoding various proteins (eg, polypeptides) or peptides.

According to various embodiments, the present invention can be used to purify mRNA synthesized in vitro of various lengths. In some embodiments, the present invention provides a length of about 1 kb, 1.5 kb, 2 kb, 2.5 kb, 3 kb, 3.5 kb, 4 kb, 4.5 kb, 5 kb, 6 kb, 7 kb, 8 kb, 9 kb, 10 kb, 11 kb, 12 kb, 13 kb, 14 kb, 15 kb or 20 kb or more can be used to purify in vitro synthesized mRNA. In some embodiments, the present invention has a length of about 1-20 kb, about 1-15 kb, about 1-10 kb, about 5-20 kb, about 5-15 kb, about 5-12 kb, about 5-10 kb kb, about 8-20 kb, or about 8-15 kb can be used to purify in vitro synthesized mRNA. For example, a typical mRNA may be about 1 kb to about 5 kb in length. More typically, the mRNA will have a length of about 1 kb to about 3 kb. However, in some embodiments, the mRNA in a composition of the invention is much longer (greater than about 20 kb).

In certain embodiments, mRNA nucleotides are modified to provide a “modified mRNA”. Thus, the modified mRNA according to the present invention may include, for example, a backbone modification, a sugar modification or a base modification. In some embodiments, antibodies encoding the mRNA (e.g., heavy and light chains encoding the mRNA) contain natural nucleotides and/or nucleotide analogs (modified nucleotides), such as, but not limited to, purines (adenine (A) , guanine (G)) or pyrimidine (thymine (T), cytosine (C), uracil (U)), modified nucleotide analogs or derivatives of purines and pyrimidines, such as 1-methyl-adenine, 2-Methyl-adenine, 2-methylthio-N-6-Isopentenyl-adenine, N6-methyl-adenine, N6-Isopentenyl-adenine, 2-thio-cytosine, 3-methyl-cytosine, 4-acetyl -Cytosine, 5-methyl-cytosine, 2,6-diaminopurine, 1-methyl-guanine, 2-methyl-guanine, 2,2-dimethyl-guanine, 7-methyl-guanine, inosine, 1-methyl-inosine , pseudouracil (5-uracil), dihydro-uracil, 2-thio-uracil, 4-thio-uracil, 5-carboxymethylaminomethyl-2-thio-uracil, 5- (carboxyhydroxymethyl)-uracil, 5-Fluoro-uracil, 5-bromo-uracil, 5-carboxymethylaminomethyl-uracil, 5-methyl-2-thio-uracil, 5-methyl-uracil, N-uracil-5-oxyacetic acid methyl ester, 5-Methylaminomethyl-uracil, 5-Methoxyaminomethyl-2-thio-uracil, 5'-methoxycarbonylmethyl-uracil, 5-methoxy-uracil, uracil-5-oxyacetic acid methyl ester, uracil- 5-oxyacetic acid (v), 1-methyl-pseudouracil, queosin, beta-D-mannosyl-queosin, wybutoxosine, phosphoramidate, phosphorothioate, peptide nucleotide, methylphospho nate, 7-deazaguanosine, 5-methylcytosine and inosine. Preparation of such analogs is described, for example, in U.S. Patent No. 4,373,071, U.S. Patent No. 4,401,796, U.S. Patent No. 4,415,732, U.S. Patent No. 4,458,066, U.S. Patent No. 4,500,707, U.S. Patent No. 4,668,777, U.S. Patent No. 4,973,679, U.S. Patent No. 5,047,524, U.S. Patent 5,132,418, U.S. Pat. No. 5,153,319, U.S. Pat. Nos. 5,262,530 and 5,700,642 to those skilled in the art, the disclosures of which are incorporated herein by reference in their entirety.

In some embodiments, the present invention can be used to purify unmodified in vitro synthesized mRNA.

In some embodiments, the mRNA comprises 5' and/or 3' untranslated regions (UTRs). In some embodiments, the 5' untranslated region comprises one or more elements that affect stability or translation of mRNA, eg, iron responsive elements. In some embodiments, the 5' untranslated region may be between about 50 and 500 nucleotides in length. In some embodiments, the 3' untranslated region comprises one or more polyadenylation signals, a binding site for a protein that affects the positional stability of mRNA in a cell, or one or more binding sites for a miRNA. In some embodiments, the 3' untranslated region may be 50 to 500 nucleotides or more in length. In some embodiments, the 5' untranslated region comprises one or more elements that affect stability or translation of mRNA, eg, iron responsive elements.

Exemplary 3' and/or 5' UTR sequences can be derived from stable mRNA molecules (e.g., globin, actin, GAPDH, tubulin, histones, and citric acid cycle enzymes) to increase the stability of the sense mRNA molecule. . For example, the 5' UTR sequence may be a partial sequence of the CMV immediate-early 1 (IE1) gene, or a fragment thereof, to improve nuclease resistance and/or improve the half-life of a polynucleotide. may include It is also contemplated to include a sequence encoding human growth hormone (hGH), or a fragment thereof, at the 3' end or untranslated region of the polynucleotide (eg, mRNA) to further stabilize the polynucleotide. . In general, these features improve the stability and/or pharmacokinetic properties (e.g., half-life) of a polynucleotide compared to the same polynucleotide lacking such features, which, e.g., for in vivo nuclease digestion Features designed to improve the resistance of polynucleotides, such as

mRNA synthesis

mRNA can be synthesized according to any of a variety of known methods. For example, mRNA can be synthesized via in vitro transcription (IVT). Although the present invention is particularly useful for purifying mRNA synthesized by an in vitro transcription reaction, it can be used in the purification of mRNA produced by any other method. In some embodiments, mRNA from other sources, including wild-type mRNA produced from bacteria, fungi, plants and/or animals, is contemplated as being within the scope of the present invention.

The presence of reagents and residual enzymes and proteins is undesirable in the final mRNA product according to several embodiments described in the previous section and may therefore be referred to as impurities or contaminants. Likewise, preparations containing one or more of these impurities or contaminants may be referred to as impure preparations. In some embodiments, in vitro transcription occurs in a single batch. In some embodiments, the IVT response comprises a capping and tailing reaction (C/T). In some embodiments, the capping and tailing reactions are performed separately from the IVT reaction. In some embodiments, recovery of the mRNA from the IVT reaction is followed by a first precipitation and purification of the mRNA by the methods described herein; The recovered purified mRNA is then capped and tailed and subjected to a second precipitation and purification.

In some embodiments, the present invention provides at least 10 mg, 50 mg, 100 mg, 200 mg, 300 mg, 400 mg, 500 mg, 600 mg, 700 mg, 800 mg, 900 mg, 1 g, 5 g, 10 g, 25 g, 50 g, 75 g, 100 g, 250 g, 500 g, 750 g, 1 kg, 5 kg, 10 kg, 50 kg, 100 kg, 1000 kg or more of an mRNA containing composition or It can be used to purify the batch. In some embodiments, the mRNA molecule is greater than 600, 700, 800, 900, 1000, 2000, 3000, 4000, 5000, 10,000 or more nucleotides in length; mRNAs of any length in between are also encompassed by the present invention.

IVT reaction

IVT is typically a promoter, a pool of ribonucleotide triphosphates, a buffer system that may include DTT and magnesium ions, and an appropriate RNA polymerase (eg, T3, T7 or SP6 RNA polymerase), DNAse I, a pyrophosphatase, and/or an RNase inhibitor containing a linear or circular DNA template. The exact conditions will vary depending on the particular application. A suitable DNA template typically has a promoter for in vitro transcription, such as a T3, T7 or SP6 promoter, followed by a desired mRNA and a desired nucleotide sequence for a termination signal. In some embodiments, the resulting mRNA is codon optimized.

In some embodiments, an exemplary IVT reaction mixture comprises a linearized double stranded DNA template with an SP6 polymerase-specific promoter, SP6 RNA polymerase, RNase inhibitor, pyrophosphatase, 29 mM NTP, 10 mM DTT and reaction buffer ( at 10x 800 mM HEPES, 20 mM _{spermidine, 250 mM MgCl 2} , at pH 7.7), and RNase-free water in a sufficient amount (QS) to the desired reaction volume; The reaction mixture is then incubated at 37° C. for 60 minutes. Then, to facilitate digestion of the double-stranded DNA template in the preparation for purification, DNase I and DNase I buffers (100 mM Tris-HCl, 5 mM MgCl ₂ and 25 mM CaCl ₂ at 10x, pH 7.6) to quench the polymerase reaction. This embodiment was shown to be sufficient to produce 100 grams of mRNA.

Other IVT methods are available in the art and may be used to practice the present invention.

Capping and Tailing (C/T) Response

Typically, mRNA processing in eukaryotes involves the addition of a "cap" on the N-terminal (5') end, and a "tail" on the C-terminal (3') end. A typical cap is a 7-methylguanosine cap, which is a guanosine linked to the first transcribed nucleotide via a 5'-5'-triphosphate bond. The presence of the cap is important in providing resistance to nucleases found in most eukaryotic cells. In some embodiments, the in vitro transcribed mRNA is prepared by addition of a 5' N ⁷ -methylguanylate cap 0 structure using a guanylate transferase, and as described in Fechter, P.; Brownlee, GG "Recognition of mRNA cap structures by viral and cellular proteins" J. Gen. Virology 2005, 86, 1239-1249] by enzymatic treatment by addition of a methyl group at the 2' O position of the second-to-last nucleotide to generate a Cap 1 structure using a 2' O-methyltransferase as described in is transformed

In some embodiments, the 5' cap is typically added as follows: first, the RNA terminal phosphatase removes one of the terminal phosphate groups from the 5' nucleotide, leaving two terminal phosphates; Guanosine triphosphate (GTP) is then added to the terminal phosphate via guanylyl transferase to create a 5'-5' triphosphate linkage; The 7-nitrogen of guanine is methylated by methyltransferase. Examples of cap structures include, but are not limited to, m7G(5')ppp(5')G, G(5')ppp(5')A, and G(5')ppp(5')G. Briefly, purified IVT mRNA is typically prepared in _{the presence of a reaction buffer comprising Tris-HCl, MgCl 2} , and RNase-free H ₂ O, GTP, S-adenosyl methionine, RNase inhibitor, 2′-Omethyl It is mixed with transferase, guanylyl transferase, and then incubated at 37°C.

In some embodiments, after addition of the Cap 1 structure, the poly-adenylate tail is added by enzymatic treatment using poly-A polymerase to the 3' end of the in vitro transcribed mRNA. The tail is typically a polyadenylation event in which a polyadenylyl moiety is added to the 3' end of the mRNA molecule. In some embodiments, after incubation for the capping reaction, the tailing reaction is initiated by adding a tailing buffer comprising _{Tris-HCl, NaCl, MgCl 2} , ATP, poly A polymerase and RNase-free H _{2 O.} The reaction is quenched by addition of EDTA.

The presence of this “tail” serves to protect the mRNA from exonuclease degradation. The 3' tail may be added before or after or concurrently with the addition of the 5' cap.

In some embodiments, the poly A tail is 25-5,000 nucleotides in length. Typically, the tail structure comprises a poly A and/or poly C tail (A, adenosine; C, cytosine). In some embodiments, the poly-A or poly-C tail on the 3' end of the mRNA is at least 50 adenosine or cytosine nucleotides, at least 150 adenosine or cytosine nucleotides, at least 200 adenosine or cytosine nucleotides, at least 250 adenosine, respectively or cytosine nucleotides, at least 300 adenosine or cytosine nucleotides, at least 350 adenosine or cytosine nucleotides, at least 400 adenosine or cytosine nucleotides, at least 450 adenosine or cytosine nucleotides, at least 500 adenosine or cytosine nucleotides, at least 550 adenosine or cytosine nucleotides, at least 600 adenosine or cytosine nucleotides, at least 650 adenosine or cytosine nucleotides, at least 700 adenosine or cytosine nucleotides, at least 750 adenosine or cytosine nucleotides, at least 800 adenosine or cytosine nucleotides, at least 850 adenosine or cytosine nucleotides, at least 900 adenosine or cytosine nucleotides, at least 950 adenosine or cytosine nucleotides, or at least 1 kb of adenosine or cytosine nucleotides. In some embodiments, the poly-A or poly-C tail comprises, respectively, about 10-800 adenosine or cytosine nucleotides (e.g., about 10-200 adenosine or cytosine nucleotides, about 10-300 adenosine or cytosine nucleotides, about 10-400 adenosine or cytosine nucleotides, about 10-500 adenosine or cytosine nucleotides, about 10-550 adenosine or cytosine nucleotides, about 10-600 adenosine or cytosine nucleotides, about 50-600 adenosine or cytosine nucleotides, about 100 to 600 adenosine or cytosine nucleotides, about 150 to 600 adenosine or cytosine nucleotides, about 200 to 600 adenosine or cytosine nucleotides, about 250 to 600 adenosine or cytosine nucleotides, about 300 to 600 adenosine or cytosine nucleotides, about 350 to 600 adenosine or cytosine nucleotides, about 400 to 600 adenosine or cytosine nucleotides, about 450 to 600 adenosine or cytosine nucleotides, about 500 to 600 adenosine or cytosine nucleotides, about 10 to 150 adenosine or cytosine nucleotides, about 10-100 adenosine or cytosine nucleotides, about 20-70 adenosine or cytosine nucleotides, or about 20-60 adenosine or cytosine nucleotides). In some embodiments, the tail structure comprises a combination of poly A and poly C tails of various lengths described herein. In some embodiments, the tail structure is at least 50%, 55%, 65%, 70%, 75%, 80%, 85%, 90%, 92%, 94%, 95%, 96%, 97%, 98%. or 99% of adenosine nucleotides. In some embodiments, the tail structure is at least 50%, 55%, 65%, 70%, 75%, 80%, 85%, 90%, 92%, 94%, 95%, 96%, 97%, 98%. or 99% of cytosine nucleotides.

Other capping and/or tailing methods are available in the art and may be used to practice the present invention.

Contaminants in mRNA synthesized in vitro

For example, mRNA products from synthetic processes as described above can contain various contaminants (also referred to as impurities), such as residual template DNA, incomplete products, enzymes such as polymerases, such as SP6 or T7-polymerases, capping enzymes. , for example guanylyl transferase, or methyl guanylyl transferase, DNase 1, various salts, and prematurely incomplete mRNA oligonucleotides that are by-products of mRNA synthesis reactions.

mRNA purification

The purification process according to the invention can be carried out during or subsequent to the synthesis. For example, mRNA can be purified as described herein before caps and/or tails are added to the mRNA. In some embodiments, mRNA is purified after caps and/or tails have been added to the mRNA. In some embodiments, mRNA is purified after cap is added. In some embodiments, mRNA is purified both before and after caps and/or tails are added to the mRNA. In general, purification steps as described herein may be performed after each step of mRNA synthesis, optionally in conjunction with other purification processes (eg, dialysis).

mRNA precipitation

In accordance with the present invention, mRNA in an impure preparation, such as an in vitro synthetic reaction mixture, can be precipitated using the buffers and suitable conditions described herein, followed by various purification methods known in the art. As used herein, the term “precipitation” (or any grammatical synonym thereof) refers to the formation of an insoluble material (eg, a solid) in solution. The term "precipitation" when used in reference to mRNA refers to the formation of an insoluble or solid form of mRNA in a liquid.

Typically, mRNA precipitation is accompanied by denaturing conditions. As used herein, the term “denaturing conditions” refers to any chemical or physical condition that can cause disruption of the native identity of an mRNA. Since the native form of a molecule is usually the most water-soluble, disrupting the secondary and tertiary structure of the molecule can cause a change in solubility and lead to precipitation of mRNA from solution.

For example, a suitable method for precipitating mRNA from an impure preparation involves treating the impure preparation with a denaturing reagent to precipitate the mRNA. Exemplary denaturing reagents suitable for the present invention include, but are not limited to, lithium chloride, sodium chloride, potassium chloride, guanidinium chloride, guanidinium thiocyanate, guanidinium isothiocyanate, ammonium acetate, and combinations thereof. . Suitable reagents may be provided in solid form or as a solution.

In some embodiments, a guanidinium salt is used in the denaturing buffer for precipitation of mRNA. As a non-limiting example, the guanidinium salt may include guanidinium chloride, guanidinium thiocyanate, or guanidinium isothiocyanate. Guanidinium thiocyanate (also called guanidine thiocyanate) can be used to precipitate mRNA. The present invention provides that, upon mRNA precipitation, a buffer comprising a guanidinium salt (eg, guanidinium thiocyanate) is used at a higher concentration than typically used for denaturation reactions, thereby substantially free of protein contaminants. It is based on the surprising discovery that it can produce mRNA. In some embodiments, a solution suitable for mRNA precipitation contains guanidine thiocyanate at a concentration greater than 4 M.

According to the present invention, in some embodiments, the buffer comprising a denaturing reagent suitable for mRNA precipitation comprises greater than 4 M guanidine thiocyanate. In some embodiments, the buffer comprising a denaturing reagent suitable for mRNA precipitation comprises about 5 M of GSCN. In some embodiments, the buffer comprising a denaturing reagent suitable for mRNA precipitation comprises about 5.5 M of GSCN. In some embodiments, the buffer comprising a denaturing reagent suitable for mRNA precipitation comprises about 6 M of GSCN. In some embodiments, the buffer comprising a denaturing reagent suitable for mRNA precipitation comprises about 6.5 M of GSCN. In some embodiments, the buffer comprising a denaturing reagent suitable for mRNA precipitation comprises about 7 M of GSCN. In some embodiments, a buffer comprising a denaturing reagent suitable for mRNA precipitation comprises about 7.5 M of GSCN. In some embodiments, the buffer comprising a denaturing reagent suitable for mRNA precipitation comprises about 8 M of GSCN. In some embodiments, the buffer comprising a denaturing reagent suitable for mRNA precipitation comprises about 8.5 M of GSCN. In some embodiments, the buffer comprising a denaturing reagent suitable for mRNA precipitation comprises about 9 M of GSCN. In some embodiments, the buffer comprising a denaturing reagent suitable for mRNA precipitation comprises about 10 M of GSCN. In some embodiments, the buffer comprising a denaturing reagent suitable for mRNA precipitation comprises greater than 10 M GSCN.

In addition to denaturing reagents, suitable solutions for mRNA precipitation may contain additional salts, surfactants and/or buffers. For example, a suitable solution may further comprise sodium lauryl sarcosyl and/or sodium citrate. In some embodiments, a suitable buffer for mRNA precipitation comprises about 5 mM sodium citrate. In some embodiments, a suitable buffer for mRNA precipitation comprises about 10 mM sodium citrate. In some embodiments, a suitable buffer for mRNA precipitation comprises about 20 mM sodium citrate. In some embodiments, a buffer suitable for mRNA precipitation comprises about 25 mM sodium citrate. In some embodiments, a buffer suitable for mRNA precipitation comprises about 30 mM sodium citrate. In some embodiments, a suitable buffer for mRNA precipitation comprises about 50 mM sodium citrate.

In some embodiments, a suitable buffer for mRNA precipitation comprises a surfactant such as N-lauryl sarcosine (sarcosyl). In some embodiments, a buffer suitable for mRNA precipitation comprises about 0.01% N-lauryl sarcosine. In some embodiments, a buffer suitable for mRNA precipitation comprises about 0.05% N-lauryl sarcosine. In some embodiments, a buffer suitable for mRNA precipitation comprises about 0.1% N-lauryl sarcosine. In some embodiments, a buffer suitable for mRNA precipitation comprises about 0.5% N-lauryl sarcosine. In some embodiments, a buffer suitable for mRNA precipitation comprises 1% N-lauryl sarcosine. In some embodiments, a buffer suitable for mRNA precipitation comprises about 1.5% N-lauryl sarcosine. In some embodiments, a buffer suitable for mRNA precipitation comprises about 2%, about 2.5%, or about 5% N-lauryl sarcosine.

In some embodiments, a solution suitable for mRNA precipitation comprises a reducing agent. In some embodiments, the reducing agent is dithiothreitol (DTT), beta-mercaptoethanol (b-ME), tris(2-carboxyethyl)phosphine (TCEP), tris(3-hydroxypropyl)phosphine ( THPP), dithioerythritol (DTE) and dithiobutylamine (DTBA). In some embodiments, the reducing agent is dithiothreitol (DTT).

In some embodiments, DTT is present at a final concentration greater than 1 mM and up to about 200 mM. In some embodiments, DTT is present at a final concentration of 2.5 mM to 100 mM. In some embodiments, DTT is present at a final concentration of 5 mM to 50 mM.

In some embodiments, DTT is present at a final concentration of 1 mM or greater. In some embodiments, DTT is present at a final concentration of 2 mM or greater. In some embodiments, DTT is present at a final concentration of 3 mM or greater. In some embodiments, DTT is present at a final concentration of 4 mM or greater. In some embodiments, DTT is present at a final concentration of 5 mM or greater. In some embodiments, DTT is present at a final concentration of 6 mM or greater. In some embodiments, DTT is present at a final concentration of 7 mM or greater. In some embodiments, DTT is present at a final concentration of 8 mM or greater. In some embodiments, DTT is present at a final concentration of 9 mM or greater. In some embodiments, DTT is present at a final concentration of 10 mM or greater. In some embodiments, DTT is present at a final concentration of 11 mM or greater. In some embodiments, DTT is present at a final concentration of 12 mM or greater. In some embodiments, DTT is present at a final concentration of 13 mM or greater. In some embodiments, DTT is present at a final concentration of 14 mM or greater. In some embodiments, DTT is present at a final concentration of 15 mM or greater. In some embodiments, DTT is present at a final concentration of 16 mM or greater. In some embodiments, DTT is present at a final concentration of 17 mM or greater. In some embodiments, DTT is present at a final concentration of 18 mM or greater. In some embodiments, DTT is present at a final concentration of 19 mM or greater. In some embodiments, DTT is present at a final concentration of about 20 mM.

In some embodiments, the denaturing buffer comprises 2 M of GSCN or greater, and DTT. In some embodiments, the denaturing buffer comprises 3 M of GSCN or greater, and DTT. In some embodiments, the denaturing buffer comprises 4 M of GSCN or greater, and DTT. In some embodiments, the denaturing buffer comprises about 5 M of GSCN or greater, and DTT. In some embodiments, the denaturing buffer comprises about 6 M of GSCN or greater, and DTT. In some embodiments, the denaturing buffer comprises about 7 M of GSCN or greater, and DTT. In some embodiments, the denaturing buffer comprises about 8 M of GSCN or greater, and DTT. In some embodiments, the denaturing buffer comprises about 9 M of GSCN or greater, and DTT.

In some embodiments, the denaturing buffer comprises 1 mM DTT or greater, and a GSCN concentration of about 5 M. In some embodiments, the denaturing buffer comprises 2 mM DTT or greater, and a GSCN concentration of about 5 M. In some embodiments, the denaturing buffer comprises 3 mM DTT or greater, and a GSCN concentration of about 5 M. In some embodiments, the denaturing buffer comprises 4 mM DTT or greater, and a GSCN concentration of about 5 M. In some embodiments, the denaturing buffer comprises 5 mM DTT or greater, and a GSCN concentration of about 5 M. In some embodiments, the denaturing buffer comprises 6 mM DTT or greater, and a GSCN concentration of about 5 M. In some embodiments, the denaturing buffer comprises 7 mM DTT or greater, and a GSCN concentration of about 5 M. In some embodiments, the denaturing buffer comprises 8 mM DTT or greater, and a GSCN concentration of about 5 M. In some embodiments, the denaturing buffer comprises 9 mM DTT or greater, and a GSCN concentration of about 5 M. In some embodiments, the denaturing buffer comprises 10 mM DTT or greater, and a GSCN concentration of about 5 M.

Protein denaturation can occur even with low concentrations of denaturing reagents in the presence or absence of reducing agents. The combination of a high concentration of GSCN with a high concentration of DTT in a denaturing solution as provided by the method of the present invention is surprising and unexpected, for example because it is a highly chaotropic solution. Unexpectedly, such a combination, when used for precipitation of impurity-containing mRNA according to the method of the present invention, surprisingly provides mRNA that is pure and substantially free of protein contamination. The mRNA precipitated in the buffer according to the method of the present invention can be processed through a filter. In some embodiments, after a single precipitation followed by filtration using a buffer comprising about 5 M of GSCN and about 10 mM DTT, the eluate is surprisingly of high quality and of high purity with no detectable protein impurities. Additionally, the method comprises, at a throughput of a wide range of mRNAs, about 1 gram, or about 10 grams, or about 100 grams, or about 500 grams, without impeding the flow of fluid through the filter, or reproducible on a scale comprising about 1000 grams of mRNA and more.

In some embodiments, the buffer for the precipitation step further comprises an alcohol. In some embodiments, the precipitation is performed under conditions wherein mRNA, a denaturing buffer (comprising GSCN and a reducing agent such as DTT) and alcohol are present in a volume ratio of 1: (5): (3). In some embodiments, the precipitation is performed under conditions wherein mRNA, denaturing buffer and alcohol are present in a volume ratio of 1: (3.5): (2.1). In some embodiments, the precipitation is performed under conditions wherein mRNA, denaturing buffer and alcohol are present in a volume ratio of 1: (4): (2). In some embodiments, the precipitation is performed under conditions wherein mRNA, denaturing buffer and alcohol are present in a volume ratio of 1: (2.8): (1.9). In some embodiments, the precipitation is performed under conditions wherein mRNA, denaturing buffer and alcohol are present in a volume ratio of 1: (2.3): (1.7). In some embodiments, precipitation is performed under conditions wherein mRNA, denaturing buffer and alcohol are present in a volume ratio of 1: (2.1): (1.5).

In some embodiments, it is desirable to incubate the impure formulation with one or more denaturing reagents described herein for a period of time at a desired temperature to allow precipitation of significant amounts of mRNA. For example, a mixture of impure agent and denaturant can be incubated for a period of time at room temperature or ambient temperature. In some embodiments, a suitable incubation time is a period of about 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 40, 50 or 60 minutes or more. In some embodiments, a suitable incubation time is a period of about 60, 55, 50, 45, 40, 35, 30, 25, 20, 15, 10, 9, 8, 7, 6 or 5 minutes or less. In some embodiments, the mixture is incubated at room temperature for about 5 minutes. Typically, “room temperature” or “ambient temperature” refers to a temperature in the range of about 20-25°C, for example about 20°C, 21°C, 22°C, 23°C, 24°C or 25°C. In some embodiments, the mixture of the impure agent and the denaturant is also prepared above room temperature (e.g., about 30-37°C, or particularly about 30°C, 31°C, 32°C, 33°C, 34°C, 35°C, at 36°C or 37°C) or below room temperature (eg, at about 15-20°C, or particularly at about 15°C, 16°C, 17°C, 18°C, 19°C or 20°C). The incubation period can be adjusted based on the incubation temperature. Typically, higher incubation temperatures require shorter incubation times.

Alternatively or additionally, a solvent may be used to facilitate mRNA precipitation. Exemplary suitable solvents include, but are not limited to, isopropyl alcohol, acetone, methyl ethyl ketone, methyl isobutyl ketone, ethanol, methanol, denatonium, and combinations thereof. For example, a solvent (eg, absolute ethanol) may be added to the impure formulation after incubation or addition of a denaturing reagent as described herein to further enhance and/or promote mRNA precipitation or with a denaturing reagent. can be Typically, after addition of a suitable solvent (eg, absolute ethanol), the mixture may be incubated at room temperature for an additional period of time. Typically, a suitable period of incubation time is about 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 40, 50 or 60 minutes or more. In some embodiments, a suitable incubation period is about 60, 55, 50, 45, 40, 35, 30, 25, 20, 15, 10, 9, 8, 7, 6 or 5 minutes or less. Typically, the mixture is incubated at room temperature for about 5 minutes. Temperatures above or below room temperature can be used with appropriate control of the incubation time. Alternatively, incubation can occur at 4°C or -20°C for precipitation.

mRNA recovery

As a result of precipitation, a suspension containing precipitated mRNA and various contaminants is formed. In some embodiments, the suspension mixture is subjected to filtration for isolation and recovery of mRNA. In some embodiments, tangential flow filtration (TFF) is used for separation of mRNA from contaminants after precipitation. For further teachings regarding TFF for mRNA purification, see USSN 14/775,915, filed September 14, 2015, to related application by the present applicant, issued May 1, 2018 as U.S. Patent 9,957,499, of the invention Title: "METHODS FOR PURIFICATION OF MESSENGER RNA"); or USSN 14/696,140, filed on April 24, 2015, issued as U.S. Patent 9,850,269 on December 26, 2017, entitled "METHODS FOR PURIFICATION OF MESSENGER RNA", or USSN 14/696,140 may be obtained from USSN 15/831,252, filed December 4, 2017, as a continuation application, the subject matter of each of which is incorporated herein by reference in its entirety. In some embodiments, normal flow filtration is performed on the precipitated mRNA and contaminants. Additional teachings on normal flow filtration (NFF) for mRNA purification can be read from US 62/722,674, filed Aug. 24, 2018, a related application by the present applicant, which is incorporated by reference in its entirety. . In some embodiments, the mRNA is recovered by centrifugation. Further teachings include USSN 15/907,086, filed Feb. 27, 2018, to related applicants, entitled "METHODS FOR PURIFICATION OF MESSENGER RNA" by Applicant; USSN 15/906,864, entitled "METHODS FOR PURIFICATION OF MESSENGER RNA", filed February 27, 2018, which encompasses purification methods using Nutsche filters; or from USSN 15/907,163, filed February 27, 2018, entitled "LARGE SCALE SYNTHESIS OF MESSENGER RNA", the respective subject matter of each of which is incorporated herein by reference in its entirety. In some embodiments, the precipitated mRNA as described in the preceding section is subjected to chromatographic separation, or any other known method in the art for recovery of pure mRNA.

In some embodiments, the methods described herein result in precipitation and recovery of significant amounts of mRNA from impure preparations. In some embodiments, the methods described herein achieve precipitation and recovery of at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% of total mRNA from the impure formulation. generate In some embodiments, the methods described herein result in precipitation and recovery of at least 80% of total mRNA from the impure formulation. In some embodiments, the methods described herein result in precipitation and recovery of substantially 90% of total mRNA from the impure formulation. In some embodiments, the methods described herein result in precipitation and recovery of substantially 91% of total mRNA from the impure formulation. In some embodiments, the methods described herein result in precipitation and recovery of substantially 92% of total mRNA from the impure formulation. In some embodiments, the methods described herein result in precipitation and recovery of substantially 93% of total mRNA from the impure formulation. In some embodiments, the methods described herein result in precipitation and recovery of substantially 94% of total mRNA from the impure formulation. In some embodiments, the methods described herein result in precipitation and recovery of substantially 95% of total mRNA from the impure formulation. In some embodiments, the methods described herein result in precipitation and recovery of substantially 96% of total mRNA from the impure formulation. In some embodiments, the methods described herein result in precipitation and recovery of substantially 97% of total mRNA from the impure formulation. In some embodiments, the methods described herein result in precipitation and recovery of substantially 98% of total mRNA from the impure formulation. In some embodiments, the methods described herein result in precipitation and recovery of substantially 99% of total mRNA from the impure formulation. In some embodiments, the methods described herein result in precipitation and recovery of substantially 100% of total mRNA from the impure formulation.

In some embodiments, mRNA purified by the methods disclosed herein is full-length mRNA determined by various detection methods described herein and known in the art (eg, capillary electrophoresis, gel electrophoresis, HPLC or UPLC). Less than 10%, less than 9%, less than 8%, less than 7%, less than 6%, less than 5%, less than 4%, less than 3%, less than 2%, less than 1%, less than 0.5%, less than 0.1% include less. Impurities include IVT contaminants such as proteins, enzymes, free nucleotides and/or "short incomplete transcripts" (shortmers). As used herein, the term “shortmer” refers to any transcript that is less than full length. In some embodiments, a "shortmer" or "incomplete transcript" is less than 100 nucleotides in length, less than 90, less than 80, less than 70, less than 60, less than 50, less than 40, less than 30, less than 20, or less than 10 nucleotides. In some embodiments, the shortmer is detected or quantified after addition of the 5'-cap and/or 3'-poly A tail.

Among other things, the purification methods described herein can be used to prepare mRNA for therapeutic use. The purity and/or integrity of purified mRNA as determined by the various characterization techniques described herein can be used as a batch release criterion. In some embodiments, the release criteria for batch production of mRNA in the manufacturing process comprises capillary electrophoretic determination of one or more of the following: purified mRNA is 5% or less, 4% or less, 3% or less, 2% or less, 1 % or less of protein contaminants, or substantially free of protein contaminants; The purified mRNA comprises no more than 5%, no more than 4%, no more than 3%, no more than 2%, no more than 1% of short unfinished RNA contaminants, or is substantially free of short unfinished RNA contaminants; The purified mRNA comprises no more than 5%, no more than 4%, no more than 3%, no more than 2%, no more than 1% of salt contaminants, or is substantially free of salt contaminants; Purified mRNA comprises at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% integrity.

In some embodiments, release criteria for batch production of mRNA in the manufacturing process comprise HPLC determination of one or more of the following: purified mRNA is 5% or less, 4% or less, 3% or less, 2% or less, 1% or less or substantially free of protein contaminants; The purified mRNA comprises no more than 5%, no more than 4%, no more than 3%, no more than 2%, no more than 1% of short unfinished RNA contaminants, or is substantially free of short unfinished RNA contaminants; The purified mRNA comprises no more than 5%, no more than 4%, no more than 3%, no more than 2%, no more than 1% of salt contaminants, or is substantially free of salt contaminants; Purified mRNA comprises at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% integrity.

Additionally, mRNA purified according to the present invention produces high yields. For example, total purified mRNA is at least about 70%, 75%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% of It is recovered in an amount that produces a yield.

According to the present invention, mRNA can be purified on a large scale. For example, at least 0.5 gram, 1 gram, 5 gram, 10 gram, 15 gram, 20 gram, 35 gram, 40 gram, 45 gram, 50 gram, 60 gram, 70 gram, 80 gram, 90 gram, 100 gram, 200 grams, 300 grams, 400 grams, 500 grams, 1 kilogram, 10 kilograms, 50 kilograms, 100 kilograms of mRNA can be purified in a single batch.

mRNA therapeutic composition

Purified mRNA according to the present invention may be delivered as naked mRNA (unpackaged) or via a delivery vehicle. As used herein, the terms “delivery vehicle”, “delivery vehicle”, “nanoparticle” or grammatical synonyms are used interchangeably.

The carrier vehicle may be formulated (when mixed with suitable excipients) in combination with one or more additional nucleic acids, carriers, targeting ligands or stabilizing reagents, or as pharmaceutical compositions. Techniques for formulating and administering drugs can be found in the latest edition of "Remington's Pharmaceutical Sciences," Mack Publishing Co., Easton, PA. A particular delivery vehicle is selected based on its ability to facilitate transfection of the nucleic acid into target cells.

According to various embodiments, suitable delivery vehicles include polymer-based carriers such as polyethyleneimine (PEI), lipid nanoparticles (LNP) and liposomes, nanoliposomes, ceramide-containing nanoliposomes, proteoliposomes, natural and synthetic-derived exos. Both natural, synthetic and semi-synthetic lamellar bodies, nanoparticles, calcium phosphor-silicate nanoparticles, calcium phosphate nanoparticles, silicon dioxide nanoparticles, nanocrystalline particulates, semiconductor nanoparticles, poly(D-arginine) , sol-gels, nanodendrimers, starch-based delivery systems, micelles, emulsions, niosomes, multi-domain-block polymers (vinyl polymers, polypropyl acrylic acid polymers, dynamic polyconjugates), dry powder formulations, plasmids , viruses, calcium phosphate nucleotides, aptamers, peptides and other vectorial tags.

In some embodiments, a suitable delivery vehicle is a liposomal delivery vehicle, eg, a lipid nanoparticle (LNP) or a liposome. In some embodiments, the liposome may comprise one or more cationic lipids. In some embodiments, the liposome comprises one or more cationic lipids, one or more non-cationic lipids, and one or more PEG-modified lipids. In some embodiments, the liposome comprises at least one or more PEG-modified lipids. In some embodiments, the liposome comprises no more than four distinct lipid components. In some embodiments, the liposome comprises no more than three distinct lipid components. In some embodiments, the one distinct lipid component is a sterol-based cationic lipid.

As used herein, the term “cationic lipid” refers to any of several lipid and lipidoid species having a net positive charge at a selected pH, such as a physiological pH. Several cationic lipids have been described in the literature, many of which are commercially available.

In some embodiments the mRNA encodes an intracellular protein or peptide. In some embodiments, the mRNA encodes a cytosolic protein. In some embodiments, the mRNA encodes a protein associated with the actin cytoskeleton. In some embodiments, the mRNA encodes a protein associated with the plasma membrane. In some specific embodiments, the mRNA encodes a transmembrane protein. In some specific embodiments the mRNA encodes an ion channel protein. In some embodiments, the mRNA encodes a perinuclear protein. In some embodiments, the mRNA encodes a nuclear protein. In some specific embodiments, the mRNA encodes a transcription factor. In some embodiments, the mRNA encodes a chaperone protein. In some embodiments, mRNA encoding the enzyme is cell (for example a mRNA, encoding an enzyme associated with a lysosomal storage urea cycle, or a metabolic disorder). In some embodiments, the mRNA encodes a protein involved in cellular metabolism, DNA repair, transcription and/or translation. In some embodiments, the mRNA encodes an extracellular protein or peptide. In some embodiments, the mRNA encodes a protein or peptide associated with the extracellular matrix. In some embodiments the mRNA encodes a secreted protein or peptide. In certain embodiments, the mRNA used in the compositions and methods of the invention may be used to express a functional peptide, protein or enzyme excreted or secreted by one or more target cells into the surrounding extracellular fluid (e.g., mRNA encoding neurotransmitters and/or hormones). In some embodiments the mRNA encodes an immunogenic protein for vaccine purposes. In some embodiments the mRNA encodes an antibody or fragment thereof. In some embodiments, the mRNA encodes a metabolic protein. In some embodiments, the mRNA encodes an enzyme. In some embodiments, the mRNA encodes a receptor protein or peptide. In some embodiments, the mRNA encodes an antigen. In some embodiments, the mRNA encodes a cancer associated antigen. In some embodiments, the mRNA encodes a vaccine.

By way of non-limiting example, mRNA may be a protein or peptide such as ABC7, ABCB3, ABCB7, ABCC7, ABCD1, AKT; AKT2, AKT3, ATF4, ALAS2, alpha galactosidase, alpha-1 protease inhibitors, APA, APC; APOA1, APOA1-Milano, APOE, anti-trypsin alpha 1, arginosuccinate synthase, ASAT; ATM; ATP7B, ATR; atropine-1; ATX3; Atxn10; ATXN2; Atxn7; ATXNl; Bax; Bcl2; BRCA1; BRCA2; carbamylphosphate synthase, CASP8, CBP (Creb-BP); CDKN2a; CFTR, CREB1, CVAP, CYP1Bl, DBA, DMD, DMPK; EGFR, EIF2B1, EIF2BA, EIF2B2, EIF2B3, EIF2B5, EIF2B4; ERBB2; ERBB3; ERBB4; erythropoietin, factor IX, factor V; Factor VII, Factor VII; Factor VIII; Factor VIIIa light chain, factor X; Factor XI (F11); Factor XII deficiency (F12, HAF); Factor XIIIA (F13Al, F13A); Factor XIIIB (F13B); FBN1, a member of the FGF receptor family; FHL3; FKRP, FXN/X25; FXR1, G6PC, G6PT, GAA, galactose-1-phosphate uridylyltransferase, GLUT2, H1Fla; HBA1; HBB; HBA2, HBB, HBD, heparan N-sulfatase, HIF; HIF3a; HLH3, HPLH2, HPLH3, Huntingtin, IDH2; IDH1, IGF receptor; IGF; IGF1R, Igf2 receptor; Igf2; Igfl receptor; Igfl; ITGB2, KIAA1596; Kras; LCRB, methylmalonyl-CoA mutase, MRP7, MUNC13-4, N-acetyl-alpha-D-glucosaminidase, NOS3, NPC1, OTC (ornithine transcarbamylase), PAH, PKHD1, PKD1, PKD2, PKD4, PKLR, PKU1, PPAR gamma; PPARalpha; PRF1, PSEN2, PSF2, PTEN; RB, Retinoschisin; RING11, SBMA/SMAXl/AR; SEC63, SERPINA1, SERPINA2, SERPINA3, SERPINA5, SERPINA6, SFTPA1, SFTPB, SFTPC, SFTPD, SLC2A, SLC7A9, SMPD1, SPTB, TAP2, TAPBP, TPSN, UNC13D, VEGF-a, VEGF-b, LR VEGF-c, VLR; and WT1.

Accordingly, in certain embodiments, the present invention provides a method for preparing a therapeutic composition comprising purified mRNA encoding a peptide or polypeptide for use in the treatment or delivery to the lung or lung cells of a subject. In certain embodiments, the present invention provides a method of making a therapeutic composition comprising purified mRNA encoding an endogenous protein that may be deficient or non-functional in a subject. In certain embodiments, the present invention provides a method of making a therapeutic composition comprising purified mRNA encoding an endogenous protein that may be deficient or non-functional in a subject.

In certain embodiments, the present invention provides a method for preparing a therapeutic composition comprising purified mRNA encoding a peptide or polypeptide for use in the treatment or delivery to the lung or lung cells of a subject. In certain embodiments, the present invention is useful in a method of making an mRNA encoding the cystic fibrosis transmembrane conduction regulator CFTR. CFTR mRNA is delivered to the lungs of a subject in need of a therapeutic composition for the treatment of cystic fibrosis. In certain embodiments, the present invention provides a method for preparing a therapeutic composition comprising purified mRNA encoding a peptide or polypeptide for use in the treatment or delivery to liver or liver cells of a subject. Such peptides and polypeptides are those associated with urea cycle disorders, those associated with lysosomal storage disorders, those associated with glycogen storage disorders, those associated with amino acid metabolism disorders, those associated with lipid metabolism or fibrotic disorders, those associated with methylmalonic acidemia , or any other metabolic disorder in which treatment or delivery to liver or liver cells with the enriched full-length mRNA provides a therapeutic benefit.

In certain embodiments, the present invention provides a method for preparing a therapeutic composition comprising purified mRNA encoding a protein associated with a urea cycle disorder. In certain embodiments, the present invention provides a method of making a therapeutic composition comprising purified mRNA encoding an ornithine transcarbamylase (OTC) protein. In certain embodiments, the present invention provides a method for preparing a therapeutic composition comprising purified mRNA encoding an arginosuccinate synthetase 1 protein. In certain embodiments, the present invention provides a method of making a therapeutic composition comprising purified mRNA encoding a carbamoyl phosphate synthetase I protein. In certain embodiments, the present invention provides a method for preparing a therapeutic composition comprising purified mRNA encoding an arginosuccinate lyase protein. In certain embodiments, the present invention provides a method for preparing a therapeutic composition comprising purified mRNA encoding an arginase protein.

In certain embodiments, the present invention provides a method for preparing a therapeutic composition comprising purified mRNA encoding a protein associated with a lysosomal storage disorder. In certain embodiments, the present invention provides a method for preparing a therapeutic composition comprising purified mRNA encoding an alpha galactosidase protein. In certain embodiments, the present invention provides a method for preparing a therapeutic composition comprising purified mRNA encoding a glucocerebrosidase protein. In certain embodiments, the present invention provides a method for preparing a therapeutic composition comprising purified mRNA encoding an iduronate-2-sulfatase protein. In certain embodiments, the present invention provides a method for preparing a therapeutic composition comprising purified mRNA encoding an iduronidase protein. In certain embodiments, the present invention provides a method of making a therapeutic composition comprising purified mRNA encoding an N-acetyl-alpha-D-glucosaminidase protein. In certain embodiments, the present invention provides a method for preparing a therapeutic composition comprising purified mRNA encoding a heparan N-sulfatase protein. In certain embodiments, the present invention provides a method for preparing a therapeutic composition comprising purified mRNA encoding a galactosamine-6 sulfatase protein. In certain embodiments, the present invention provides a method for preparing a therapeutic composition comprising purified mRNA encoding a beta-galactosidase protein. In certain embodiments, the present invention provides a method for preparing a therapeutic composition comprising purified mRNA encoding a lysosomal lipase protein. In certain embodiments, the present invention provides a method for preparing a therapeutic composition comprising purified mRNA encoding an arylsulfatase B (N-acetylgalactosamine-4-sulfatase) protein. In certain embodiments, the present invention provides a method for preparing a therapeutic composition comprising purified mRNA encoding transcription factor EB (TFEB).

In certain embodiments, the present invention provides a method for preparing a therapeutic composition comprising purified mRNA encoding a protein associated with a glycogen storage disorder. In certain embodiments, the present invention provides a method of making a therapeutic composition comprising purified mRNA encoding an acid alpha-glucosidase protein. In certain embodiments, the present invention provides a method for preparing a therapeutic composition comprising purified mRNA encoding a glucose-6-phosphatase (G6PC) protein. In certain embodiments, the present invention provides a method of making a therapeutic composition comprising purified mRNA encoding a liver glycogen phosphorylase protein. In certain embodiments, the present invention provides a method for preparing a therapeutic composition comprising purified mRNA encoding a muscle phosphoglycerate mutase protein. In certain embodiments, the present invention provides a method for preparing a therapeutic composition comprising purified mRNA encoding a glycogen debranching enzyme.

In certain embodiments, the present invention provides a method for preparing a therapeutic composition comprising purified mRNA encoding a protein associated with amino acid metabolism. In certain embodiments, the present invention provides a method for preparing a therapeutic composition comprising purified mRNA encoding a phenylalanine hydroxylase enzyme. In certain embodiments, the present invention provides a method for preparing a therapeutic composition comprising purified mRNA encoding a glutaryl-CoA dehydrogenase enzyme. In certain embodiments, the present invention provides a method for preparing a therapeutic composition comprising purified mRNA encoding a propionyl-CoA carboxylase enzyme. In certain embodiments, the present invention provides a method for preparing a therapeutic composition comprising purified mRNA encoding an oxalase alanine-glyoxylate aminotransferase enzyme.

In certain embodiments, the present invention provides a method for preparing a therapeutic composition comprising purified mRNA encoding a protein associated with a lipid metabolism or fibrotic disorder. In certain embodiments, the present invention provides a method of making a therapeutic composition comprising purified mRNA encoding an mTOR inhibitor. In certain embodiments, the present invention provides a method for preparing a therapeutic composition comprising purified mRNA encoding an ATPase phospholipid transporter 8B1 (ATP8B1) protein. In certain embodiments, the present invention provides a method encoding one or more of one or more NF-kappa B inhibitors, such as I-kappa B alpha, interferon-related developmental regulator 1 (IFRD1), and sirtuin 1 (SIRT1). A method for preparing a therapeutic composition comprising purified mRNA is provided. In certain embodiments, the present invention provides a method for preparing a therapeutic composition comprising purified mRNA encoding a PPAR-gamma protein or an active variant.

In certain embodiments, the present invention provides a method for preparing a therapeutic composition comprising purified mRNA encoding a protein associated with methylmalonic acidemia. For example, in certain embodiments, the present invention provides a method of making a therapeutic composition comprising purified mRNA encoding a methylmalonyl CoA mutase protein. In certain embodiments, the present invention provides a method for preparing a therapeutic composition comprising purified mRNA encoding a methylmalonyl CoA epimerase protein.

In certain embodiments, the present invention provides a method of making a therapeutic composition comprising purified mRNA wherein treatment of or delivery to the liver may provide a therapeutic benefit. In certain embodiments, the present invention provides a method for preparing a therapeutic composition comprising purified mRNA encoding an ATP7B protein (also known as a Wilson disease protein). In certain embodiments, the present invention provides a method for preparing a therapeutic composition comprising purified mRNA encoding a porphobilinogen deaminase enzyme. In certain embodiments, the present invention provides a method for preparing a therapeutic composition comprising purified mRNA encoding one or more coagulation enzymes, such as Factor VIII, Factor IX, Factor VII and Factor X. In certain embodiments, the present invention provides a method for preparing a therapeutic composition comprising purified mRNA encoding a human hemochromatosis (HFE) protein.

In certain embodiments, the present invention provides a method for preparing a therapeutic composition comprising purified mRNA encoding a peptide or polypeptide for use in the treatment or delivery of a cardiovascular disease or cardiovascular cell in a subject. In certain embodiments, the present invention provides a method for preparing a therapeutic composition comprising purified mRNA encoding vascular endothelial growth factor A protein. In certain embodiments, the present invention provides a method for preparing a therapeutic composition comprising purified mRNA encoding a relaxin protein. In certain embodiments, the present invention provides a method for preparing a therapeutic composition comprising purified mRNA encoding a bone morphogenetic protein-9 protein. In certain embodiments, the present invention provides a method for preparing a therapeutic composition comprising purified mRNA encoding a bone morphogenetic protein-2 receptor protein.

In certain embodiments, the present invention provides a method for preparing a therapeutic composition comprising purified mRNA encoding a peptide or polypeptide for use in the treatment or delivery to muscle or muscle cells of a subject. In certain embodiments, the present invention provides a method for preparing a therapeutic composition comprising purified mRNA encoding a dystrophin protein. In certain embodiments, the present invention provides a method for preparing a therapeutic composition comprising purified mRNA encoding a prataxin protein. In certain embodiments the present invention provides a method for preparing a therapeutic composition comprising purified mRNA encoding a peptide or polypeptide for use in the treatment or delivery to cardiac muscle or cardiac muscle cells of a subject. In certain embodiments, the present invention provides a method of making a therapeutic composition comprising purified mRNA encoding a protein that modulates one or both of potassium and sodium channels in muscle tissue or muscle cells. In certain embodiments, the present invention provides a method for preparing a therapeutic composition comprising purified mRNA encoding a protein that modulates a Kv7.1 channel in muscle tissue or muscle cells. In certain embodiments, the present invention provides a method for preparing a therapeutic composition comprising purified mRNA encoding a protein that modulates Nav1.5 channels in muscle tissue or muscle cells.

In certain embodiments, the present invention provides a method for preparing a therapeutic composition comprising purified mRNA encoding a peptide or polypeptide for use in the treatment or delivery to the nervous system or cells of the nervous system in a subject. For example, in certain embodiments, the present invention provides a method of making a therapeutic composition comprising purified mRNA encoding a survival motor neuron 1 protein. For example, in certain embodiments, the present invention provides a method of making a therapeutic composition comprising purified mRNA encoding a survival motor neuron 2 protein. In certain embodiments, the present invention provides a method for preparing a therapeutic composition comprising purified mRNA encoding a prataxin protein. In certain embodiments, the invention provides a method of making a therapeutic composition comprising purified mRNA encoding an ATP binding cassette subclass D member 1 (ABCD1) protein. In certain embodiments, the present invention provides a method for preparing a therapeutic composition comprising purified mRNA encoding a CLN3 protein.

In certain embodiments, the present invention provides a method for preparing a therapeutic composition comprising purified mRNA encoding a peptide or polypeptide for use in the treatment or delivery to the blood or bone marrow or blood or bone marrow cells of a subject. In certain embodiments, the present invention provides a method for preparing a therapeutic composition comprising purified mRNA encoding beta globin protein. In certain embodiments, the present invention provides a method for preparing a therapeutic composition comprising purified mRNA encoding a Bruton's tyrosine kinase protein. In certain embodiments, the present invention provides a method for preparing a therapeutic composition comprising purified mRNA encoding one or more coagulation enzymes, such as Factor VIII, Factor IX, Factor VII and Factor X.

In certain embodiments, the present invention provides a method for preparing a therapeutic composition comprising purified mRNA encoding a peptide or polypeptide for use in the treatment or delivery to the kidney or kidney cells of a subject. In certain embodiments, the present invention provides a method for preparing a therapeutic composition comprising purified mRNA encoding a collagen type IV alpha 5 chain (COL4A5) protein.

In certain embodiments, the present invention provides a method for preparing a therapeutic composition comprising purified mRNA encoding a peptide or polypeptide for use in the treatment or delivery to the eye or ocular cells of a subject. In certain embodiments, the present invention provides a method of making a therapeutic composition comprising purified mRNA encoding an ATP-binding cassette subclass A member 4 (ABCA4) protein. In certain embodiments, the present invention provides a method for preparing a therapeutic composition comprising purified mRNA encoding a retinoskicin protein. In certain embodiments, the present invention provides a method for preparing a therapeutic composition comprising purified mRNA encoding a retinal pigment epithelium-specific 65 kDa (RPE65) protein. In certain embodiments, the present invention provides a method for preparing a therapeutic composition comprising purified mRNA encoding a 290 kDa centrosome protein (CEP290).

In certain embodiments, the present invention provides a method for preparing a therapeutic composition comprising purified mRNA encoding a peptide or polypeptide for use in treatment with or delivery of a vaccine to a subject or cells of a subject. For example, in certain embodiments the present invention provides a method of making a therapeutic composition comprising purified mRNA encoding an antigen from an infectious agent such as a virus. In certain embodiments, the present invention provides a method for preparing a therapeutic composition comprising purified mRNA encoding an antigen from an influenza virus. In certain embodiments, the present invention provides a method for preparing a therapeutic composition comprising purified mRNA encoding an antigen from a respiratory syncytial virus. In certain embodiments, the present invention provides a method for preparing a therapeutic composition comprising purified mRNA encoding an antigen from a rabies virus. In certain embodiments, the present invention provides a method for preparing a therapeutic composition comprising purified mRNA encoding an antigen from a cytomegalovirus. In certain embodiments, the present invention provides a method for preparing a therapeutic composition comprising purified mRNA encoding an antigen from rotavirus. In certain embodiments, the present invention provides a method for preparing a therapeutic composition comprising purified mRNA encoding an antigen from a hepatitis virus, such as hepatitis A virus, hepatitis B virus or hepatitis C virus. In certain embodiments, the present invention provides a method for preparing a therapeutic composition comprising purified mRNA encoding an antigen from human papillomavirus. In certain embodiments, the present invention provides a method for preparing a therapeutic composition comprising purified mRNA encoding an antigen from a herpes simplex virus, such as herpes simplex virus 1 or herpes simplex virus 2. In certain embodiments, the present invention provides a method for preparing a therapeutic composition comprising purified mRNA encoding an antigen from a human immunodeficiency virus, such as human immunodeficiency virus type 1 or human immunodeficiency virus type 2. In certain embodiments, the present invention provides a method for preparing a therapeutic composition comprising purified mRNA encoding an antigen from human metapneumovirus. In certain embodiments, the invention provides a therapeutic composition comprising purified mRNA encoding an antigen from a human parainfluenza virus, such as human parainfluenza virus type 1, human parainfluenza virus type 2, or human parainfluenza virus type 3 A manufacturing method is provided. In certain embodiments, the present invention provides a method for preparing a therapeutic composition comprising purified mRNA encoding an antigen from a malaria virus. In certain embodiments, the present invention provides a method for preparing a therapeutic composition comprising purified mRNA encoding an antigen from Zika virus. In certain embodiments, the present invention provides a method for preparing a therapeutic composition comprising purified mRNA encoding an antigen from a chikungunia virus.

In certain embodiments, the present invention provides a method of making a therapeutic composition comprising purified mRNA that encodes an antigen identified from cancer cells of a subject or associated with cancer in a subject. In certain embodiments, the present invention provides a method for preparing a therapeutic composition comprising purified mRNA encoding an antigen determined from a subject's own cancer cells (ie, for providing a personalized cancer vaccine). In certain embodiments, the present invention provides a method for preparing a therapeutic composition comprising purified mRNA encoding an antigen expressed from a mutant KRAS gene.

In certain embodiments, the invention provides a method of making a therapeutic composition comprising purified mRNA encoding an antibody. In certain embodiments, the antibody may be a bispecific antibody. In certain embodiments, the antibody may be part of a fusion protein. In certain embodiments, the present invention provides a method for preparing a therapeutic composition comprising purified mRNA encoding an antibody to OX40. In certain embodiments, the present invention provides a method for preparing a therapeutic composition comprising purified mRNA encoding an antibody to VEGF. In certain embodiments, the present invention provides a method for preparing a therapeutic composition comprising purified mRNA encoding an antibody to tissue necrosis factor alpha. In certain embodiments, the present invention provides a method for preparing a therapeutic composition comprising purified mRNA encoding an antibody to CD3. In certain embodiments, the present invention provides a method for preparing a therapeutic composition comprising purified mRNA encoding an antibody to CD19.

In certain embodiments, the present invention provides a method of making a therapeutic composition comprising purified mRNA encoding an immunomodulatory agent. In certain embodiments, the present invention provides a method for preparing a therapeutic composition comprising purified mRNA encoding interleukin 12. In certain embodiments, the present invention provides a method for preparing a therapeutic composition comprising purified mRNA encoding interleukin 23. In certain embodiments, the present invention provides a method for preparing a therapeutic composition comprising purified mRNA encoding interleukin 36 gamma. In certain embodiments, the present invention provides a method for preparing a therapeutic composition comprising purified mRNA encoding a constitutively active variant of one or more stimulators of the interferon gene (STING) protein.

In certain embodiments, the present invention provides a method for preparing a therapeutic composition comprising purified mRNA encoding an endonuclease. In certain embodiments, the present invention provides a method of making a therapeutic composition comprising purified mRNA encoding an RNA-guided DNA endonuclease protein, such as a Cas 9 protein. In certain embodiments, the present invention provides a method for preparing a therapeutic composition comprising purified mRNA encoding a meganuclease protein. In certain embodiments, the present invention provides a method for preparing a therapeutic composition comprising purified mRNA encoding a transcriptional activator-like effector nuclease protein. In certain embodiments, the present invention provides a method for preparing a therapeutic composition comprising purified mRNA encoding a zinc finger nuclease protein.

In certain embodiments, the present invention provides a method for preparing a therapeutic composition comprising purified mRNA encoding for the treatment of an ocular disease. In some embodiments the method is used for the preparation of a therapeutic composition comprising purified mRNA encoding retinoskicin.

Another aspect of the present invention is a purified mRNA composition prepared by an aspect or embodiment described above.

In some embodiments the present invention provides a pharmaceutical composition comprising a solution comprising the purified mRNA of the above description and at least one pharmaceutically acceptable excipient.

Typically, suitable mRNA solutions may also contain buffers and/or salts. In general, buffers may include HEPES, ammonium sulfate, sodium bicarbonate, sodium citrate, sodium acetate, potassium phosphate and sodium phosphate.

Pharmaceutically acceptable salts are well known in the art. For example, SM Berge et al. [J. Pharmaceutical Sciences (1977) 66:1-19 describes in detail pharmaceutically acceptable salts. Pharmaceutically acceptable salts of the compounds of the present invention include those derived from suitable inorganic and organic acids and bases. Examples of pharmaceutically acceptable non-toxic acid addition salts include the use of inorganic acids (such as hydrochloric acid, hydrobromic acid, phosphoric acid, sulfuric acid and perchloric acid) or organic acids (such as acetic acid, oxalic acid, maleic acid, tartaric acid, citric acid, succinic acid or malonic acid) or other methods used in the art (eg, ion exchange). Other pharmaceutically acceptable salts include adipate, alginate, ascorbate, aspartate, benzenesulfonate, benzoate, bisulfate, borate, butyrate, camphorate, camphorsulfonate, citrate, cyclopentanepro. Cypionate, digluconate, dodecylsulfate, ethanesulfonate, formate, fumarate, glucoheptonate, glycerophosphate, gluconate, hemisulphate, heptanoate, hexanoate, hydroiodide, 2 -Hydroxy-ethanesulfonate, lactobionate, lactate, laurate, lauryl sulfate, maleate, maleate, malonate, methanesulfonate, 2-naphthalenesulfonate, nicotinate, nitrate, ole ate, oxalate, palmitate, palmoate, pectinate, persulfate, 3-phenylpropionate, phosphate, picrate, pivalate, propionate, stearate, succinate, sulfate, tartrate, thiocyanate nates, p-toluenesulfonates, undecanoates, valerate salts, and the like. Salts derived from suitable bases include alkali metal, alkaline earth metal, ammonium and N ⁺ (C _1-4 alkyl) ₄ salts. Representative alkali or alkaline earth metal salts include sodium, lithium, potassium, calcium, magnesium, and the like. Further pharmaceutically acceptable salts include, where appropriate, non-toxic ammonium, quaternary ammonium formed using counterions such as halides, hydroxides, carboxylates, sulfates, phosphates, nitrates, sulfonates and aryl sulfonates. , and amine cations. Additional pharmaceutically acceptable salts include salts formed from quaternization of amines with appropriate electrophiles (eg, alkyl halides) to form quaternized alkylated amino salts.

One aspect of the present invention is a method of treating a disease or disorder comprising administering to a subject in need thereof the pharmaceutical composition of the above aspect.

Another aspect of the invention is a solution comprising the purified mRNA prepared by the aspect or embodiment described above.

Any aspect or embodiment described herein may be combined with any other aspect or embodiment as disclosed herein. While the disclosure has been described in conjunction with its detailed description, the foregoing description is intended to illustrate and not to limit the scope of the disclosure as defined by the scope of the appended claims. Other aspects, advantages and modifications are within the scope of the following claims.

Further teachings related to the present invention are described in WO 2010/053572; WO 2011/068810; WO 2012/075040; WO 2012/170889; WO 2012/170930; WO 2013/063468; WO 2013/149140; WO 2013/149141; WO 2013/185067; WO 2013/185069; WO 2014/089486; WO 2014/152513; WO 2014/152659; WO 2014/152673; WO 2014/152774; WO 2014/152966; WO 2014/153052; WO 2015/061461; WO 2015/061467; WO 2015/061491; WO 2015/061500; WO 2015/148247; WO 2015/164773; WO 2015/184256; WO 2015/200465; WO 2016/004318; WO 2016/149508; WO/2014/152940; PCT/US16/57044; US 62/320,073; US 62/349,331; US 62/420,413; US 62/420,421; US 62/420,428; US 62/420,435; US 62/421,007; US 62/421,021, each of which is incorporated herein by reference in its entirety.

Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, suitable methods and materials are described below. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. References cited herein are not admitted to be prior art to the claimed invention. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting.

<Example>

Example 1. Improved mRNA Purification Using 5 M GSCN Precipitation Buffer

In this example, 5 M GSCN precipitation buffer was compared to a buffer with 4 M GSCN for its ability to remove process enzymes on the milligram scale using a column filtration format. The purified product obtained after a single precipitation step followed by filtration preferably has reduced or undetectable protein contaminants as determined by silver stained gel analysis.

Materials and Methods

CFTR mRNA was prepared by in vitro transcription as described elsewhere. Briefly, an exemplary IVT reaction was performed with a linear or circular DNA template comprising a codon-optimized CFTR sequence, the template DNA also comprising a promoter, a pool of ribonucleotide triphosphates, a buffer system comprising DTT and magnesium ions, and an appropriate RNA polymerase (eg, T3, T7 or SP6 RNA polymerase), DNAse I, pyrophosphatase, and/or RNAse inhibitor.

A reaction involving the incorporation of a 5'-cap and a 3' poly A tail (cap-tail reaction) was performed immediately prior to purification. The 5' cap was added as follows: first adding a terminal phosphatase to the reaction, wherein the RNA terminal phosphatase removes one of the terminal phosphate groups from the 5' nucleotide, leaving two terminal phosphates; adding guanosine triphosphate (GTP) via guanylyl transferase to the terminal phosphate to create a 5'-5' triphosphate linkage; The 7-nitrogen of guanine was then methylated by methyltransferase. The construct was further processed to incorporate poly (A) tails using poly (A) polymerase. Then, the reaction mixture comprising mRNA was subjected to precipitation and subsequent steps as described below. In Table 1 , the materials used in the method steps described in this example are summarized.

Table 1

Precipitation : A 2.5 mg CFTR mRNA cap-tail reaction was performed using one of the precipitation buffers listed above (4 M GSCN control, 5 M GSCN 1% or 5 M GSCN 0.1% N-lauryl sarcosine) under the following conditions. Precipitation: 1 volume of mRNA reaction was mixed with 2.3 volumes of precipitation buffer and mixed thoroughly. For each volume of the initial cap tail reaction, 1.7 volumes of 100% EtOH were added to RNA:precipitation buffer and mixed thoroughly to precipitate.

Filtration : The precipitated mixture was loaded onto a maxiprep filtration column (Qiagen, Germantown, MD) and centrifuged to capture the precipitated mRNA onto a Qiagen filter (all centrifugation). was performed at 3,600 RPM). The captured mRNA precipitate was then washed twice with 80% EtOH (1 mL of 80% EtOH for 1 mg of RNA per wash) by centrifugation of 2 min after the first wash and 10 min of centrifugation after the second wash. did. Elution was performed by adding water to the membrane followed by incubation at room temperature and centrifugation. This process was repeated a total of 5 times with a total elution volume of 2.0 mL. The absorbance at 260 nm was measured and the concentration was determined for each sample.

Silver Staining : A silver staining gel was run using the method described in the preceding examples. 15.5 μl of purified RNA at 1 mg/ml was treated with 4 μl of RNaseI (100 U/mL, Invitrogen) at 37° C. for 30 minutes. Samples were prepared in Invitrogen LDS loading buffer with reducing reagent and loaded onto a 10% Bis-Tris gel. Electrophoresis was performed at 200 V for 35 minutes. Gels were stained for 8 minutes using a SilverQuest staining kit.

result

After a single purification cycle of the CFTR mRNA captail reaction as described above, the yield for each sample was calculated using Nano-Drop to determine the concentration (A260 nm), which is shown in Table 2 (below). summarized. mRNA yield = elution concentration (mg/mL) * elution volume (mL). Yields from all conditions were acceptable and were within the range of desired yields based on the scale of the reaction.

Table 2

Samples were analyzed with a silver stained gel assay to detect residual process enzymes. The silver-stained gel image is shown in FIG. 1 . 5 M GSCN-1% lauryl sarcosine buffer ( FIG. 1 , lane 6 of gel 2), or 5 M GSCN when compared to samples precipitated using 4 M GSCN buffer ( FIG. 1 , lane 5 of gel 1), or 5 M GSCN For samples precipitated using -0.1% lauryl sarcosine buffer ( FIG. 1 , lane 8 of gel 2), a significant improvement in process enzyme removal was noted. From these data, it was found that increasing GSCN molarity improved process enzyme removal, regardless of the concentration of surfactant in the buffer.

Example 2. Improved mRNA Purification Using 5 M GSCN Precipitation Buffer Containing DTT

In this example, a precipitation buffer with dithiothreitol (DTT) and a precipitation buffer without dithiothreitol (DTT) were used to remove residual enzymes (i.e. process enzymes) associated with the mRNA preparation/production process; and their ability to reduce the number of precipitations required to ensure their proper removal. mRNA purification was performed on the milligram scale by filtration using a multi-well microplate format.

Materials and Methods

CFTR mRNA was prepared by in vitro transcription as described above. Purification using the method described below for mRNA cap-tail reactions was performed. In Table 3 , the materials used in the method steps described in this example are summarized.

Table 3

Precipitation : For each well of a 24-well plate, 2.0 mg of mRNA cap-tail (C/T) reaction was performed with one of the precipitation buffers listed above (5 M GSCN, or 5 M GSCN with 10 mM DTT). was used to precipitate under the following conditions: 1 volume of mRNA reaction was added to 2.3 volumes of precipitation buffer and mixed thoroughly. For each 1 volume of the initial cap-tail reaction, 1.7 volumes of 100% EtOH was added to RNA:precipitation buffer and mixed thoroughly to precipitate.

Filtration : The precipitated mixture was loaded into individual wells of a 24-well filter plate, and a vacuum pressure of about 5 PSI was applied to the plate to capture the precipitated mRNA. The captured mRNA precipitate was then washed twice with 80% EtOH by 5 PSI vacuum filtration after the first and second wash (1 mL of 80% EtOH for 1 mg RNA per wash). Elution was effected by addition of water to the membrane followed by incubation at room temperature and vacuum filtration. This process was repeated a total of 5 times with a total elution volume of 2.0 mL per well. The absorbance at 260 nm was measured and the concentration was determined for each sample.

Silver Staining : Silver staining gels were run with the following pre-stained sample preparations using an Invitrogen kit (Invitrogen, Carlsbad, CA) according to the manufacturer's instructions. 15.5 μl of purified RNA at 1 mg/ml was treated with 4 μl of RNaseI (100 U/mL, Invitrogen) at 37° C. for 30 minutes. Samples were prepared in Invitrogen LDS loading buffer with reducing reagent and loaded onto a 10% Bis-Tris gel. Electrophoresis was performed at 200 V for 35 minutes. The gel was stained for 8 minutes using the SilverQuest silver staining kit (Invitrogen, Carlsbad, CA).

result

After a single purification of the CFTR mRNA cap-tail reaction as described above, the total yield for each sample was measured for mRNA elution using a nano-drop spectrophotometer (Nano Drop Technologies, Wilmington, Del.). After measuring the optical density, it was calculated to determine the mRNA concentration (A260 nm). mRNA yield = elution concentration (mg/mL) * elution volume (mL). The results are summarized in Table 4 below. In Table 4, the desired yields from the DTT buffer-based protocol are specified for the reaction scale.

Table 4

Samples were analyzed in a silver staining assay as described in the preceding paragraph to detect silver residual process enzymes. The silver-stained gel image is shown in FIG. 2 . Lane 6 of gel 1 ( FIG. 2 , left image) corresponds to a sample purified using 5 M GSCN without DTT. Lane 6 of gel 2 ( FIG. 2 , right image) corresponds to a sample purified using 5 M GSCN with 10 mM DTT. When compared to 5 M GSCN alone, for samples precipitated using 5 M GSCN with 10 mM DTT buffer (compare gel 1 lane 6 and gel 2 lane 6), a significant improvement was noted in the removal of the process enzyme . Lane 1 in either gel 1 or gel 2 represents a control sample showing a faint band of contaminant enzyme. Lanes 10-12 in each gel represent electrophoretic transfer controls of the process enzymes RNAse1 (lane 10), SP6 (lane 11), guanylate transferase (lane 12), respectively.

Example 3. Purification of 1 gram capped and tailed mRNA using GSCN-DTT precipitation buffer

In this example, the ability of GSCN-DTT buffer to successfully remove detectable levels of contaminant residual enzymes from gram-scale mRNA production processes was evaluated.

Materials and Methods

mRNA was processed on a batch purification scale of 1 gram. CFTR mRNA was synthesized using the in vitro transcription method described above. Capping and tailing reactions were performed immediately prior to purification. Table 5 summarizes the materials used in the process described in this example.

Table 5

Precipitation : 1 gram (1G) of CFTR mRNA capping and tailing reaction (cap-tail reaction) was precipitated using 5 M GSCN with DTT under the following conditions. One volume of mRNA reaction was added to 2.3 volumes of precipitation buffer and mixed thoroughly. For 1 volume of the initial cap-tail reaction, 1.7 volumes of 100% EtOH was added to the mRNA:precipitation buffer solution and mixed thoroughly to precipitate. After precipitation, 10 g of Solka-Floc 100 was added to act as a filter agent (filter aid) to prevent membrane fouling and aid in final elution.

Filtration : The precipitated mixture with filter agent was loaded onto a 1 L vacuum filtration flask and vacuum was applied to filter the precipitate out of the precipitation buffer. The captured mRNA precipitate was then washed with 80% EtOH (1 L of 80% EtOH per g RNA) followed by a 10-minute vacuum drying step. Elution was performed by removing the precipitated mRNA and filter aid and suspending in 250 mL of water. The suspension was stirred at room temperature for 30 min and then passed through a fresh 0.22 μm PES filter flask to remove filter aid. The absorbance at 260 nm was measured and the concentration was determined for each sample.

Silver Staining : The silver staining gel was run with the following pre-stained sample preparations according to the Invitrogen kit. 15.5 μl of RNA at 1 mg/ml was treated with 4 μl of RNaseI (100 U/mL, Invitrogen) at 37° C. for 30 minutes. Samples were prepared in Invitrogen LDS loading buffer with reducing reagent, which were ultimately loaded onto a 10% Bis-Tris gel. Electrophoresis was performed at 200 V for 35 minutes. The gel was stained using the SilverQuest staining kit and allowed to run for 8 minutes.

Result :

After one round of purification of the CFTR mRNA cap tail reaction as described above, the sample yield was calculated using nano-drops to determine the concentration (A260 nm), which is summarized in Table 6 (below). The mRNA yields from the 1G reaction were acceptable and were within the range of the desired yields based on the scale of the reaction.

Table 6

Samples were analyzed with a silver stained gel to detect the presence of any residual enzyme from the process. The silver stained gel image is shown in Figure 3 below. No observable residual enzyme was detected in the purified CFTR mRNA sample ( FIG. 3 , lane 5). Electrophoretic transfer controls for process enzymes are shown on the gel as follows: RNAse1 (lane 6), SP6 (lane 7), guanylate transferase (lane 8), poly A polymerase (lane 9) and 2 -O-methyl transferase (lane 10). These results demonstrate that gram-scale cap-tail mRNA was successfully precipitated, captured, washed and recovered using a 1 L 0.22 μm vacuum filter flask with filter aid and 5 M GSCN-10 mM DTT. Surprisingly, in a single purification step, any detectable level of residual process enzyme could be removed based on the sensitivity of our detection method.

Example 4. Purification of 10 grams of capped and tailed mRNA using GSCN-DTT precipitation buffer

In this example, the ability of GSCN-DTT buffer to successfully remove detectable levels of contaminant residual enzymes from mRNAs processed on a 10 gram batch purification scale with a single precipitation step was evaluated.

Materials and Methods:

CFTR mRNA was synthesized using the in vitro transcription method described above. Capping and tailing reactions were performed immediately prior to purification. Table 7 summarizes the materials used in the process described below.

Table 7

Precipitation : 10 grams (10 G) of CFTR mRNA capping and tailing reaction (cap-tail reaction) was precipitated using 5 M GSCN w/DTT under the following conditions. One volume of mRNA reaction was added to 2.3 volumes of precipitation buffer and mixed thoroughly. For 1 volume of the initial cap-tail reaction, 1.7 volumes of 100% EtOH was added to the RNA:precipitation buffer solution and mixed thoroughly to precipitate. After precipitation, 20 g of Solka-Floc 100 was added to act as a filter agent to prevent membrane fouling and aid in final dissolution.

Filtration : The precipitated mixture with filter agent was loaded onto a H300P filtration centrifuge (Heinkel USA, Swedenboro, NJ) at 1500 RPM at 2.0 L/min. The captured RNA precipitate was then washed with 80% EtOH (5 L of 80% EtOH per gram of RNA) followed by a drying step of 15 minutes. Elution was performed by removing the precipitated mRNA and filter aid and suspending in 5 L of water. The suspension was stirred at room temperature for 30 min and then passed through a H300P filtration centrifuge to remove filter aid. Two additional 5 L elutions were performed to recover as much material as possible from the filter aid cake. The absorbance at 260 nm was measured and the concentration was determined for each sample.

Silver Staining : A silver stained agarose gel was run with the following pre-stained sample preparations according to the Invitrogen kit. 15.5 μl of RNA at 1 mg/ml was treated with 4 μl of RNaseI (100 U/mL, Invitrogen) at 37° C. for 30 minutes. Samples were prepared in Invitrogen LDS loading buffer with reducing reagent, which were ultimately loaded onto a 10% Bis-Tris gel. Electrophoresis was performed at 200 V for 35 minutes. The gel was stained using the SilverQuest staining kit and allowed to run for 8 minutes.

Capillary Electrophoresis (CE) : RNA integrity and tail length were assessed using a CE fragment analyzer and large RNA detection kit. Analysis of the peak profile for size shift and completeness with respect to tail length was performed.

result:

After a single purification of the CFTR mRNA cap tail reaction as described above, the sample yield was calculated using nano-drops to determine the concentration (A260 nm), which is summarized in Table 8 below. The final mRNA yield from the 10 G reaction was good and was within the range of the desired yield based on the scale of the reaction.

Table 8

Samples were analyzed with a silver stained gel assay to detect residual process enzymes. The silver stained gel image is shown in FIG. 4 , which shows that no residual process enzyme is observable in the purified CFTR mRNA sample (lane 4). Lane 1 shows a control purified mRNA sample showing a faint band of contaminant enzyme. Lanes 7-11 depict electrophoretic transfer controls for the following process enzymes: RNAse1 (lane 7), SP6 (lane 8), guanylate transferase (lane 9), poly A polymerase (lane 10) and 2-O-methyl transferase (lane 11).

RNA integrity and tail length after single purification using H300P and 5 M GSCN w/10 mM DTT were assessed by CE. The integrity of the mRNA after a single purification is demonstrated in Figures 5A and 5B. Figure 5A shows the mRNA peak before the tailing reaction. Figure 5B shows the mRNA peak after tailing reaction of the same native mRNA preparation. A single distinct peak for the CE electrophoresis in FIG. 5B is consistent with historical control samples (not shown). A poly-A tail length of 534 nt was evaluated based on CE gel migration compared to untailed RNA ( FIG. 5B versus FIG. 5A ). The tail length was within the predicted tail size based on the tailing reaction conditions.

From the examples, it was shown that 10 G of mRNA was successfully precipitated, captured, washed and recovered using a H300P centrifuge using 5 M GSCN-10 mM DTT as precipitation buffer. In a single purification process using 5 M GSCN-10 mM DTT, any detectable level of residual process enzyme can be removed based on the sensitivity of our detection method. Filter centrifugation with 5 M GSCN-10 mM DTT did not negatively affect mRNA integrity or tails when compared to past samples.

Example 5. Purification of 100 grams of capped and tailed mRNA using GSCN-DTT precipitation buffer

In this example, the ability of GSCN-DTT buffer to successfully remove detectable levels of contaminant residual enzyme from mRNA that was treated at a batch purification scale of 100 grams (100 G) with a single precipitation step was evaluated.

Materials and Methods:

CFTR mRNA was synthesized using the in vitro transcription method described above. Capping and tailing reactions were performed immediately prior to purification. Table 9 summarizes the materials used in the process described below.

Table 9

Precipitation : 100 G of CFTR mRNA cap-tail reaction was precipitated using 5 M GSCN with DTT under the following conditions. One volume of mRNA reaction was added to 2.3 volumes of precipitation buffer and mixed thoroughly. For 1 volume of the initial cap-tail reaction, 1.7 volumes of 100% EtOH was added to the RNA:precipitation buffer solution and mixed thoroughly to precipitate. After precipitation, 1200 g of Solka-Floc 100 was added to act as a filter agent to prevent membrane fouling and aid in final dissolution.

Filtration : The precipitated mixture with filter agent was loaded onto an EHBL503 filtration centrifuge (Rousselet Robatel, Anone, France) at 1000 RPM at 6.0 L/min. The captured RNA precipitate was then washed with 80% EtOH (1.5 L of 80% EtOH per gram of RNA) followed by a 20 min drying step. Elution was performed by removing the precipitated mRNA and filter aid and suspending in 25 L of water. The suspension was stirred at room temperature for 30 minutes and then passed through an EHBL503 filtration centrifuge to remove filter aid. Two additional 15 L elutions were performed to recover as much material as possible from the filter aid cake. The absorbance at 260 nm was measured and the concentration was determined for each sample.

Silver Staining : The silver staining gel was run with the following pre-stained sample preparations according to the Invitrogen kit. 15.5 μl of RNA at 1 mg/ml was treated with 4 μl of RNaseI (100 U/mL, Invitrogen) at 37° C. for 30 minutes. Samples were prepared in Invitrogen LDS loading buffer with reducing reagent, which were ultimately loaded onto a 10% Bis-Tris gel. Electrophoresis was performed at 200 V for 35 minutes. The gel was stained using the SilverQuest staining kit and allowed to run for 8 minutes.

CE : RNA integrity and tail length were assessed using a CE fragment analyzer and large RNA detection kit. Analysis of the peak profile for size shift and completeness with respect to tail length was performed.

Cap assay : The cap species present in the final purified mRNA product was determined using the HPLC/MS method, allowing the accurate calculation of uncapped, CapG, Cap0 and Cap1 amounts, which were calculated based on the total signal. Reported as a percentage.

result:

After a single purification of the CFTR mRNA cap tail reaction as described above, the sample yield was calculated using nano-drops to determine the concentration (A260 nm), which is summarized in Table 10 below. The final mRNA yield from the 100 G reaction was within the range of the desired yield based on the scale of the reaction.

Table 10

A silver stained gel image is shown in FIG. 6 , from which it was found that there was no observable residual process enzyme in the purified CFTR mRNA sample (lanes 5 and 6). It is surprising that a single purification step using the buffer of the present invention can produce high purity mRNA free of detectable contaminant processing enzyme(s). In this figure, lanes 8-12 are the following lanes RNAse1 (lane 8), SP6 (lane 9), guanylate transferase (lane 10), poly A polymerase (lane 11) and 2-O-methyl transferase ( Electrophoretic transfer controls for process enzymes such as lane 12) are shown.

CE electrophoresis of RNA shows a single distinct peak consistent with a representative historical control sample ( FIG. 7 , indicated by arrows). A poly-A tail length of 300 nt was evaluated relative to uncapped RNA (data not shown) based on CE gel migration in C/T mRNA ( FIG. 7 ). The tail length was within the predicted tail size based on the tailing reaction conditions.

The percentage of Cap1 species in the final mRNA product after a single purification with 5 M GSCN w/10 mM DTT was (95%). All other species were uncapped (5%). These results were consistent with the historical cap 1% for this transcript (data not shown).

From these data, it was found that 100 g of mRNA was successfully precipitated, captured, washed and recovered using an EHBL503 centrifuge with 5 M GSCN-10 mM DTT as precipitation buffer. In a single purification process using 5 M GSCN-10 mM DTT, any detectable level of residual process enzyme can be removed based on the sensitivity of our detection method. Using a filtration centrifuge with 5 M GSCN-10 mM DTT did not adversely affect mRNA integrity, tail length, mRNA capping efficiency or stability when compared to past samples.

Example 6. Purification of capped and tailed mRNA using various reducing agents

In this example, various reducing reagents were evaluated for their ability to remove process enzymes when added to 5 M GSCN precipitation buffer.

Materials and Methods:

Eight batches of 5 mg of CFTR mRNA were synthesized using the in vitro transcription method described above. For precipitation, 2.3 volumes of 5 M GSCN buffer (2.3 M GSCN final) with various reducing agents listed in Table 11 were added per volume of IVT reaction. To the mixture was then added 1.7 volumes of 100% EtOH (final 34% EtOH). The precipitated RNA sample was captured on a Qiagen RNeasy Maxi column, washed twice with 10 mL of 80% ethanol and then dissolved in 2 mL of _{RNase-free H 2 O.} The concentration of the lysed RNA sample was calculated using the absorbance at 260 nm using a Nano-Drop 2000 spectrophotometer.

Table 11

The resulting CFTR IVT mRNA samples were then used as starting material for eight 5 mg cap and tail reactions. After capping and tailing, each of the 8 reactions was precipitated by adding 2.3 volumes of 5 M GSCN buffer listed in Table 11 (2.3 M GSCN final) per 1 volume of IVT reaction. To the mixture was then added 1.7 volumes of 100% EtOH (final 34% EtOH). The precipitated RNA sample was captured on a Qiagen RNeasy Maxi column, washed twice with 10 mL of 80% ethanol and then dissolved in 5 mL of _{RNase-free H 2 O.} The concentration of the lysed RNA sample was calculated using the absorbance at 260 nm using a Nano-Drop 2000 spectrophotometer.

Each sample was evaluated using a silver staining method as described in Example 1 to determine the amount of residual process enzyme present after purification with various reducing reagents.

result:

As shown in Figure 8 , use of any of the reducing agents tested at concentrations of 5-15 mM in 5 M GSCN precipitation buffer resulted in complete removal of detectable levels of RNA processing enzymes (lane 3- 9). The purified CFTR reaction (lane 2) using only 5 M GSCN without any reducing agent was hazy and RNA polymerase and GuaT in a range of molecular weights were observed, further evidence that GSCN alone is ineffective in removing RNA synthesis process enzymes. provides

These results demonstrate that when GSCN is used as the disordering salt, it is necessary to add a reducing agent in the RNA precipitation buffer to ensure adequate process enzyme removal. Interestingly, several reducing reagents at various concentrations perform similarly to the originally evaluated DTT reagent at 10 mM. While not wishing to be bound by theory, reducing the RNA processing enzyme prior to addition of an organic solvent to precipitate the RNA is an important step since the reduction step is a process when the RNA is purified using ethanol salt induced precipitation. This is because it facilitates the removal of the enzyme.

equivalent

Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. It is not intended that the scope of the invention be limited to the above description, but rather as set forth in the claims which follow.

Claims (38)

A method for removing impurities from a messenger RNA (mRNA) preparation synthesized by a large-scale in vitro transcription process (IVT), comprising:
precipitating the IVT-synthesized mRNA in a buffer containing a denaturing salt together with a reducing agent;
capturing the precipitated mRNA; and
purifying the mRNA by dissolving the captured mRNA into a solution
How to include. The method of claim 1 , further comprising capping and tailing the mRNA prior to precipitating the mRNA. The method according to claim 1, wherein the impurities in the mRNA preparation obtained by the large-scale IVT process are proteins including enzymes used for IVT, capping and tailing; incomplete RNA transcript; nucleotides and salts. The method according to claim 1, wherein the purified mRNA obtained by the method exceeds at least 90% of the amount of the synthesized mRNA. The method of claim 1 , wherein the purified mRNA obtained by the method is substantially free of any enzymatic impurities. The method of claim 1 , wherein at least about 1 mg of mRNA is synthesized in the IVT reaction. The method of claim 1 , wherein at least about 10 G of mRNA is synthesized in the IVT reaction. The method of claim 1 , wherein at least about 100 G of mRNA is synthesized in the IVT reaction. The method of claim 1 , wherein at least about 1 Kg of mRNA is synthesized in the IVT reaction. The method of claim 1 , wherein the buffer for the precipitation step further comprises an alcohol. The method according to claim 1, wherein the precipitation is carried out under conditions in which mRNA, denaturing buffer and alcohol are present in a volume ratio of 1: (5): (3). The method according to claim 1, wherein the precipitation is carried out under conditions wherein mRNA, denaturing buffer and alcohol are present in a volume ratio of 1: (3.5): (2.1). The method according to claim 1, wherein the precipitation is carried out under conditions wherein mRNA, denaturing buffer and alcohol are present in a volume ratio of 1: (2.8): (1.9). The method according to claim 1, wherein the precipitation is carried out under conditions wherein mRNA, denaturing buffer and alcohol are present in a volume ratio of 1: (2.3): (1.7). The method according to claim 1, wherein the precipitation is carried out under conditions wherein mRNA, denaturing buffer and alcohol are present in a volume ratio of 1: (2.1): (1.5). The method of claim 1 , wherein the modified salt is guanidinium thiocyanate. The method of claim 1 , wherein the guanidinium thiocyanate is present in a final concentration of at least 2 M, at least 3 M, at least 4 M, at least 5 M, at least 6 M, or at least 7 M. The method of claim 1 , wherein the guanidinium thiocyanate is present in a final concentration of at least greater than 3 M. The method of claim 1 , wherein the guanidinium thiocyanate is present in a final concentration of at least greater than 4 M. The method of claim 1 , wherein the guanidinium thiocyanate is present in a final concentration of 5 M. The method of claim 1, wherein the reducing agent is dithiothreitol (DTT), beta-mercaptoethanol (b-ME), tris(2-carboxyethyl)phosphine (TCEP), tris(3-hydroxypropyl)phosphine (THPP), dithioerythritol (DTE) and dithiobutylamine (DTBA). The method of claim 1 , wherein the reducing agent is dithiothreitol (DTT). 23. The method of any one of claims 1-22, wherein the DTT is present at a final concentration greater than 1 mM and up to about 200 mM. 24. The method according to any one of claims 1 to 23, wherein the DTT is present in a final concentration of 2.5 mM to 100 mM. 25. The method of any one of claims 1-24, wherein the DTT is present in a final concentration of 5 mM to 50 mM. The method of claim 1 , wherein the concentration of DTT is 5 mM, 10 mM, 15 mM or 20 mM. The method of claim 1 , wherein the step of precipitating the mRNA is performed more than once. The method according to claim 1, wherein the step of precipitating the mRNA is performed only once. The method of claim 1 , wherein the step of capturing the mRNA further comprises performing filtration, centrifugation, reversible adsorption to a solid phase, or a combination thereof on the mRNA. 30. The method of any one of claims 1-29, wherein the filtration is tangential flow filtration (TFF). 31. The method according to any one of claims 1 to 30, wherein the filtration is depth filtration (DF). 32. The method according to any one of claims 1 to 31, wherein the filtration is performed using a Nutsche filter. 33. The method according to any one of claims 1 to 32, wherein the filtration is performed in combination with centrifugation. The method according to claim 1, wherein the step of lysing the captured mRNA is performed in an aqueous solution. The method according to claim 1, wherein the step of the captured mRNA is further subjected to dialysis in a suitable buffer comprising a lower salt concentration. 36. The method of any one of claims 1-35, wherein the substantially free of any protein impurities is less than 5%, less than 4%, less than 3%, less than 2%, less than 1% protein impurities, or 0% protein. How impurity. 37. The method of any one of claims 1-36, wherein the purified mRNA is substantially free of salts. 38. The method of any one of claims 1-37, wherein the purified mRNA is at least greater than 95%, greater than 96%, greater than 97%, greater than 98%, greater than 99%, or about 100% of the full length encoding the protein. A method comprising mRNA.

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